Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Moment of induction and duration of experimental varicocele in rats: effects on semen quality.

PURPOSE: Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. MATERIALS AND METHODS: Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). RESULTS: The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. CONCLUSION: In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm.

Uncommon opsin's retinal isomer is involved in mammalian sperm thermotaxis.

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts-tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

A Key regulatory protein QRICH2 governing sperm function with profound antioxidant properties, enhancing sperm viability.

Infertility poses a global health and social challenge, affecting approximately 15% of couples at childbearing age, with half of the cases attributed to male factors, wherein genetic factors exert a substantial role. In our prior investigation, we identified loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to various morphological abnormalities in sperm flagella and male infertility. Moreover, our observations in Qrich2 knockout mice revealed a pronounced reduction in spermatozoa count. However, the underlying mechanism remains elusive, prompting further investigation in the current study. By conducting experiments such as Hematoxylin-eosin (HE) staining, immunofluorescence staining, flow cytometry, and single sperm metabolism analysis on the testes and spermatozoa of Qrich2 knockout mice, we found a strong antioxidant capacity mediated by QRICH2 both in vivo and in vitro. Qrich2 knockout led to elevated levels of ROS, consequently inducing DNA damage in spermatids, which in turn triggered increased autophagy and apoptosis, ultimately causing a significant decrease in spermatozoa count. Incubation with the N-terminal purified protein of QRICH2 exhibited potent strong antioxidant activity at the cell and spermatozoa levels in vitro, thereby enhancing spermatozoa viability and motility. Therefore, QRICH2 plays a crucial role in safeguarding spermatids from excessive ROS-induced damage by augmenting antioxidant capacity, thereby promoting spermatozoa survival and improving motility. Furthermore, the N-terminal purified protein of QRICH2 shows promise as an additive for protecting spermatozoa during preservation and cryopreservation.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Impact of various cryo-preservation steps on sperm rheotaxis and sperm kinematics in bull.

Semen cryopreservation is an important tool that has massively contributed to the progression of animal reproduction, especially in cattle. Nonetheless, a large part of the sperm population suffers from cryostress and loses fertility during the process. Although bovine semen cryopreservation is more advanced than any other species, there are still some missing links in the technology knowledge. The aim of the current study was to detect the effect of cryopreservation steps on sperm rheotaxis. Semen samples were collected from sex bulls and analyzed inside a microfluidic platform with CASA after each step of cryopreservation, including control, dilution with yolk citrate, cryoprotectant addition, and cooling or freezing. The results showed that positive rheotaxis % (PR) was not affected during cryopreservation. On the contrary, the sperm kinematics of the positive rheotactic sperm undergo significant changes, as velocity parameters (VCL, VSL, and VAP) were lower in both the cryoprotectant adding and cooling/freezing steps than in the control and yolk citrate dilution steps, while progression parameters (LIN and BCF) were higher in the cryoprotectant and cooling/freezing steps than in the control and yolk citrate dilution steps. Beside these results, an interesting phenomenon of sperm backward positive rheotaxis has been observed. The results of backward sperm rheotaxis samples revealed a significant decrease in PR%, while all sperm kinematics except BCF were significantly higher than normal rheotaxis samples. Based on these results, we conclude that positive rheotactic sperm cells are the elite of the sperm population; however, they still get some sublethal cryodamage, as shown by alterations in sperm kinematics. We also suggest that the sperm-positive rheotaxis mechanism is a mixture of an active and passive process rather than a passive physical one.

Clustering of spermatozoa examined through flow cytometry provides more information than the conventional assessment: a resilience to osmotic stress example.

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Exosomes from uterine fluid promote capacitation of human sperm.

Background: Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. Methods: EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). Results: The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Conclusion: Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.

Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

Sperm binding to oviduct epithelial spheroids varies among males and ejaculates but not among females in pigs.

The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150-200 µm in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 ± 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology.

Per capita sperm metabolism is density dependent.

From bacteria to metazoans, higher density populations have lower per capita metabolic rates than lower density populations. The negative covariance between population density and metabolic rate is thought to represent a form of adaptive metabolic plasticity. A relationship between density and metabolism was actually first noted 100 years ago, and was focused on spermatozoa; even then, it was postulated that adaptive plasticity drove this pattern. Since then, contemporary studies of sperm metabolism specifically assume that sperm concentration has no effect on metabolism and that sperm metabolic rates show no adaptive plasticity. We did a systematic review to estimate the relationship between sperm aerobic metabolism and sperm concentration, for 198 estimates spanning 49 species, from protostomes to humans from 88 studies. We found strong evidence that per capita metabolic rates are concentration dependent: both within and among species, sperm have lower metabolisms in dense ejaculates, but increase their metabolism when diluted. On average, a 10-fold decrease in sperm concentration increased per capita metabolic rate by 35%. Metabolic plasticity in sperm appears to be an adaptive response, whereby sperm maximize their chances of encountering eggs.

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

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Change in the swimming pattern of Salmo salar spermatozoa caused by the high temperature of the sperm motility activation medium.

Fish are ectotherms and many have an external reproductive mode. An environmental factor which triggers fish reproductive activity in fish is water temperature. However, climate change is causing increasingly frequent events in which the water temperature varies rapidly; as a result, both in hatchery and in natural conditions, fish sperm are exposed to varying environmental temperatures during their journey toward the egg. This study was based on two experiments: The first experiment was designed to determine how storage at 4 °C for four days affected the sperm functions of Atlantic salmon (Salmo salar) sperm collected by either abdominal massage (stripping/Pure) or testicular dissection (testicular macerate/Macerated). Further, computer-assisted semen analysis (CASA) was used to compare sperm velocity parameters (VCL, VSL, and VAP) and progressivity (STR, LIN, and WOB) after motility activation at different temperatures (8 and 16 °C) of sperm collected by both methods (Pure vs Macerated). The results show that spermatozoa from Macerated samples maintained a higher sperm function when stored at 4 °C for 4 days compared to Pure sperm samples. In the second experiment, CASA determined that all parameters for sperm velocity (VCL, VSL, and VAP) and progressivity (STR (50%/55%), LIN (25%-32%), and WOB (51%-57%) were affected by activation temperature (P < 0.05) and that the motility patterns after activation at 16 °C (P < 0.05), specifically the LIN or STR swimming trajectories of the sperm differed between the two groups. In conclusion, the sperm quality of testicular Macerate was superior to that of Pure sperm abdominal mass, based on the higher quality of various sperm functions during short-term storage. Moreover, there was a significant effect of the temperature of the activation medium on sperm speed and progressivity (motility pattern) in the collected samples of testicular macerate. The sensitivity of Salmo salar spermatozoa to elevated temperature varies markedly between collection methods (Pure and Macerated).

Decoding the influence of semen collection processes on goat sperm quality from a perspective of seminal plasma proteomics.

This study aims to assess the impact of semen collection methods on goat semen quality and seminal plasma (SP) proteomes. Semen was collected by artificial vagina (AV) or electro-ejaculator (EE) and semen parameters were evaluated. Tandem mass tag coupled with liquid chromatography-tandem mass spectrometry was used to screen SP differentially abundant proteins (DAPs) between EE and AV. PRM was used to confirm the reliability of the data. In contrast to EE, a lower volume, higher progressive motility and concentration were observed in AV. No differences were found in total motility, membrane integrity, acrosome integrity, and ROS production between EE and AV. In total, 1692 proteins were identified in SP, including 210 DAPs. Among them, 120 and 90 proteins were down-regulated and up-regulated in AV compared to EE, respectively. The GO annotation showed that DAPs are mainly localized in the membrane, involved in deference responses to bacterium, RNA processing, and related to oxidoreductase activity. KEGG demonstrated tight associations of DAPs with specific amino acids, carbon metabolism, citrate cycle, and propanoate metabolism. In conclusion, this study provides valuable insights into the effects of semen collection on goat semen quality and explores the potential action mechanism based on the modification of SP proteomes. SIGNIFICANCE OF THE STUDY: The quality of fresh semen directly influences the results of artificial insemination and semen cryopreservation in livestock. This study represents the first attempt to evaluate the impact of semen collection methods including electroejaculation and artificial vagina on sperm quality and seminal plasma proteomes in goat. The results of this study demonstrated that semen collection methods directly impacted the quality of goat semen. Then, the proteomic strategy was used to explore the potential action mechanism of semen collection methods on sperm. Some differentially abundant proteins that potentially influence semen quality were identified. Furthermore, this study suggests the possibility of utilizing specific proteins as predictive markers for goat semen quality.

Changes in Sperm Parameters with Time in Men with Normal and Abnormal Baseline Semen Analysis.

The association between paternal age and sperm quality in the population level has been previously studied. Only limited data exists regarding the intra-personal variations in semen parameters among fertile and infertile men over time. We aimed to assess trends over time in semen parameters among men with normal and abnormal baseline sperm parameters and investigate potential risk factors for sperm quality deterioration. This retrospective cohort study was conducted at a university-affiliated medical center in vitro fertilization (IVF) unit. Patients with at least two semen analyses (SA) performed > 1 year apart, with the last SA done between 2017 and 2021, were included. The study consisted of two main analyses-comparison of intra-patient's sperm parameters changes in men with normal and abnormal baseline SA (BSA) and analysis of risk factors for developing abnormal semen parameters over time in men who had normal BSA parameters. This study included a total of 902 men assessed for infertility with normal and abnormal BSA. The average time interval between tests was 1015 days (range 366-7709 days). Among individuals with normal BSA, there was a mild decline in most parameters-concentration (- 6.53 M/ml), motility (- 7.74%), and total motile count (TMC) (- 21.80 M) (p < 0.05 for all parameters). In contrast, a slight improvement in most parameters, except for concentration, was noted in men with abnormal BSA-volume (+ 0.21 ml), motility (+ 8.72%), and TMC (+ 14.38 M) (p < 0.05 for all parameters). Focusing on men with normal BSA, 33.5% of individuals developed abnormality in one or more of their sperm parameters over time, within a mean time of 1013 ± 661 days. We also found that only time between tests emerged as an independent prognostic factor for the development of abnormal SA later. Interestingly, sperm deterioration in participants in their third, fourth, and fifth decades of life with normal initial semen analysis was similar. Our study provides evidence of a decline in semen quality over time in individuals with normal BSA, in contrast to men with abnormal BSA. Longer time intervals between tests independently increase the risk of sperm abnormalities.

Application of sperm motion kinematics and motility-related proteins for prediction of male fertility.

The selection of superior sires is paramount for enhancing the efficiency of animal production in the livestock industry. However, semen quality assessment still relies on conventional semen analysis techniques in both animals and humans. Despite extensive efforts to develop various biomarkers for more accurate and precise predictions of male fertility potential, more effective physiological indicators and advance potential biomarkers are needed. Herein, we aimed to develop new potential biomarkers related to sperm motion kinematics for male fertility prediction. We first evaluated sperm motion kinematic parameters and expression levels of sperm motility-related proteins of 30 Duroc boars. We then explored the correlation between litter size, sperm motion kinematics parameters, and sperm motility-related proteins. Progressive sperm motility (%), rapid sperm motility (%), slow sperm motility (%), straight-line velocity (µm/s), linearity (%), beat cross frequency (Hz), mean angular displacement (degree), wobble (%) were correlated with litter size. Furthermore, the expression of axonemal dynein light intermediate polypeptide 1 (DNALI1) and radial spoke head protein 9 homolog (RSPH9) correlated with litter size. The overall accuracy exceeded 60% for predicting litter size using these sperm motion parameters and proteins. Notably, our study observed an increase in litter size after predicting litter size using these parameters and proteins. Thus, sperm motion kinematic parameters and protein expression, particularly of DNALI1 and RSPH9, could serve as new biomarkers for male fertility. These results may contribute to improved understanding of the mechanisms underlying sperm motility.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Deletion of the Sodium Glucose Cotransporter 1 (Sglt-1) impairs mouse sperm movement.

The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.

Effect of Maca aqueous extract addition to a freezing extender for canine semen.

This study investigated the effect of the addition of Lepidium meyenii (Maca) to the freezing extender on the post-thaw quality of dog semen. Ten canine ejaculates were frozen following a two-step protocol using a tris-glucose-citrate egg yolk extender with or without the addition of 10 µl/mL of aqueous extract of Maca (Maca and ctrl groups, respectively). Prior to (fresh semen) and after freezing (T0) sperm motility, kinetic parameters, viability and mitochondrial membrane potential (MMP), as well as the levels of malondialdehyde (MDA) were evaluated. In addition, sperm motility, kinetic parameters, viability and MMP were examined up to 2 h of incubation of 37 °C after thawing (T1 and T2) to evaluate thermo-resistance. The addition of Maca reduced MDA concentration at T0 (p < 0.05) and increased total motility, the percentage of sperm with medium velocity and WOB at T1. Progressive motility decreased (p < 0.05) at T1 in the ctrl group, whereas it was not affected in Maca group at any time point. In addition, the percentage of hyperactivated spermatozoa remained constant at T1 in the ctrl, while in the Maca group an increase (p < 0.05) of this parameter was recorded. Although no differences were found for MMP between groups at any time points, a decrease of viable sperm with low MMP was observed in ctrl group between T0 and T1 and in Maca group between T1 and T2. The addition of Maca prior freezing reduced the extent of lipid peroxidation and activated canine sperm motility and hyperactivation after thawing.