Showing papers on "Sperm motility published in 2018"

Rheotaxis-based separation of sperm with progressive motility using a microfluidic corral system

Abstract: The separation of motile sperm from semen samples is sought after for medical infertility treatments. In this work, we demonstrate a high-throughput microfluidic device that can passively isolate motile sperm within corrals inside a fluid channel, separating them from the rest of the diluted sample. Using finite element method simulations and proposing a model for sperm motion, we investigated how flow rate can provide a rheotaxis zone in front of the corral for sperm to move upstream/downstream depending on their motility. Using three different flow rates that provided shear rates above the minimum value within the rheotaxis zone, we experimentally tested the device with human and bovine semen. By taking advantage of the rheotactic behavior of sperm, this microfluidic device is able to corral motile sperm with progressive velocities in the range of 48-93 μm⋅s-1 and 51-82 μm⋅s-1 for bovine and human samples, respectively. More importantly, we demonstrate that the separated fractions of both human and bovine samples feature 100% normal progressive motility. Furthermore, by extracting the sperm swimming distribution within the rheotaxis zone and sperm velocity distribution inside the corral, we show that the minimum velocity of the corralled sperm can be adjusted by changing the flow rate; that is, we are able to control the motility of the separated sample. This microfluidic device is simple to use, is robust, and has a high throughput compared with traditional methods of motile sperm separation, fulfilling the needs for sperm sample preparation for medical treatments, clinical applications, and fundamental studies.

Outer dense fibers stabilize the axoneme to maintain sperm motility.

Abstract: Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non-motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down-regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α-tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α-tubulin and protect the acetylation level of α-tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α-tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo-expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.

SpermQ–A Simple Analysis Software to Comprehensively Study Flagellar Beating and Sperm Steering

Abstract: Motile cilia, also called flagella, are found across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g., in the brain or lung. For sperm, the picture has emerged that the flagellum is not only a motor but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum navigates sperm through the complex environment in the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behavior of sperm. It has been proposed that distinct signaling domains in the flagellum control the flagellar beat. However, a detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signaling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the beat pattern, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are only a little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signaling components in sperm and distinct flagellar beat patterns.

Influence of bull age, ejaculate number, and season of collection on semen production and sperm motility parameters in Holstein Friesian bulls in a commercial artificial insemination centre.

Abstract: In the current era of genomic selection, there is an increased demand to collect semen from genomically selected sires at a young age. The objective of this study was to assess the effect of bull age, ejaculate number, and season of collection on semen production (ejaculate volume, sperm concentration, and total sperm number; TSN) and sperm motility (prefreeze and post-thaw total and gross motility) parameters in Holstein Friesian bulls in a commercial artificial insemination (AI) center. The study involved the interrogation of a large dataset collected over a 4-yr period, (n = 8,983 ejaculates; n = 176 Holstein Friesian bulls aged between 9 mo and 8 yr). Bulls aged less than 1 yr had the poorest semen production and sperm motility values for all parameters assessed compared with bulls older than 1 yr (P < 0.01). First ejaculates had greater semen production and greater prefreeze motility values than second consecutive ejaculates (P < 0.01), but despite this, there was no difference in post-thaw motility. When subsequent ejaculates were collected from bulls aged less than 1 yr, semen production and sperm motility did not differ compared with mature bulls. Semen collected in winter was poorest in terms of sperm concentration and TSN, but best in terms of post-thaw motility (P < 0.01). In conclusion, second ejaculates can be collected, particularly from bulls aged less than 1 yr, without a significant decrease in post-thaw sperm motility, thus may be a useful strategy to increase semen availability from young genomically selected AI bulls in high demand.

Sperm DNA damage has a negative effect on early embryonic development following in vitro fertilization.

Abstract: Sperm DNA damage is recognized as an important biomarker of male infertility. To investigate this, sperm DNA damage was assessed by the sperm chromatin dispersion (SCD) test in semen and motile spermatozoa harvested by combined density gradient centrifugation (DGC) and swim-up in 161 couples undergoing in vitro fertilization (IVF). Semen analysis and sperm DNA damage results were compared between couples who did or did not achieve pregnancy. The sperm DNA damage level was significantly different between the two groups (P < 0.05) and was negatively correlated with IVF outcomes. Logistic regression analysis confirmed that it was an independent predictor for achieving clinical pregnancy. The effects of different levels of sperm DNA damage on IVF outcomes were also compared. There were significant differences in day 3 embryo quality, blastocyst formation rate, and implantation and pregnancy rates (P < 0.05), but not in the basic fertilization rate between the two groups. Thus, sperm DNA damage as measured by the SCD appears useful for predicting the clinical pregnancy rate following IVF.

Massive Weight Loss Obtained by Bariatric Surgery Affects Semen Quality in Morbid Male Obesity: a Preliminary Prospective Double-Armed Study

Abstract: The aim of this study is to evaluate the effect of massive weight loss on the seminal parameters at 6 months from bariatric surgery. Two-armed prospective study performed in 31 morbidly obese men, undergoing laparoscopic roux-en-Y-gastric bypass (n = 23) or non-operated (n = 8), assessing sex hormones, conventional (sperm motility, morphology, number, semen volume), and non-conventional (DNA fragmentation and seminal interleukin-8), semen parameters, at baseline and after 6 months from surgery or patients’ recruitment. In operated patients only, a statistically significant improvement in the sex hormones was confirmed. Similarly, a positive trend in the progressive/total sperm motility and number was observed, though only the increase in semen volume and viability was statistically significant (Δ = 0.6 ml and 10%, P < 0.05, respectively). A decrease in the seminal interleukin-8 levels and in the sperm DNA fragmentation was also present after bariatric surgery, whereas these parameters even increased in non-operated subjects. Age-adjusted multivariate analysis showed that the BMI variations significantly correlated with the changes in the sperm morphology (β = −0.675, P = 0.025), sperm number (β = 0.891, P = 0.000), and semen volume (r = 0.618, P = 0.015). The massive weight loss obtained with bariatric surgery was associated with an improvement in some semen parameters. The correlations found between weight loss and semen parameter variations after surgery suggest that these might occur early downstream of the testis and more slowly than the changes in the sex hormones.

Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish.

Abstract: N6-methyladenosine (m6A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic messenger RNA (mRNA). Previous studies have implicated m6A modification in multiple biological processes, but the in vivo function of m6A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Zmettl3m/m , in zebrafish. We find that the oocytes in Zmettl3m/m adult females are stalled in early development and the ratio of full-grown stage (FG) follicles is significantly lower than that of wild type. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo In Zmettl3m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Zmettl3m/m , and defective gamete maturation is accompanied by decreased overall m6A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Zmettl3m/m Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m6A modifications are essential for optimal reproduction in vertebrates.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility

Abstract: Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.

Lead exposure reduces sperm quality and DNA integrity in mice.

Abstract: Toxicity of lead on male reproductive functions has raised wide public concern as environmental lead contamination remains common worldwide. Conflicting and controversial data are available regarding effects of lead on male fertility. More importantly, our knowledge on effects of lead on sperm DNA integrity is significantly limited. Thus, further studies should focus on this issue. In the current study, adult male mice were exposed to a series of lead acetate concentrations in drinking water for six weeks. Following administration, lead levels in blood, testicles, and epididymis were measured, and potential changes in morphology of testis and epididymis due to lead exposure were identified. We also analyzed sperm parameters, including sperm density, viability, motility, and morphology, to evaluate quality of sperm collected from epididymis. Especially, hypothetical influence of lead on sperm DNA integrity was also evaluated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, alkaline comet assay, and sperm chromatin structure assay. Lead exposure possibly exerted no effect on growth of mice because these animals acquired similar body weight gain during the experimental period. However, high lead concentrations (0.5% and 1%) in drinking water affected sperm motility and increased percentage of spermatozoa with abnormal morphology. In groups treated with 0.25%, 0.5%, and 1% lead acetate, percentages of sperm cells showing DNA breaks and chromatin structure damage significantly increased. Altogether, lead exposure not only exhibits adverse effects on sperm physiological parameters, but also impairs DNA structure and integrity. These effects may lead to significant decline in male fertility.

Experimental heatwaves negatively impact sperm quality in the zebra finch.

Abstract: For sexually reproducing species, functionally competent sperm are critical to reproduction. While high atmospheric temperatures are known to influence the timing of breeding, incubation and reproductive success in birds, the effect of temperature on sperm quality remains largely unexplored. Here, we experimentally investigated the impact of ecologically relevant extreme temperatures on cloacal temperature and sperm morphology and motility in zebra finches Taeniopygia guttata We periodically sampled males exposed to 30°C or 40°C temperatures daily for 14 consecutive days. Following a 12-day (23°C) recovery period, birds were again exposed to heat, but under the alternate treatment (e.g. birds initially exposed to 40°C were exposed to 30°C). Elevated temperatures led to an increase in cloacal temperature and a reduction in the proportion of sperm with normal morphology; these effects were most notable under 40°C conditions, and were influenced by the duration of heat exposure and prior exposure to high temperature. Our findings highlight the potential role of temperature in determining male fertility in birds, and perhaps also in constraining the timing of avian breeding. Given the increased frequency of heatwaves in a warming world, our results suggest the need for further work on climatic influences on sperm quality and male fertility.

Clinical assessment of the male fertility

Abstract: The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

Abstract: STUDY QUESTIONS Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? SUMMARY ANSWER OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. WHAT IS KNOWN ALREADY Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. STUDY DESIGN, SIZE, DURATION Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity. PARTICIPANTS/MATERIALS, SETTING, METHODS Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP. MAIN RESULTS AND THE ROLE OF CHANCE TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction. LARGE SCALE DATA None. LIMITATIONS REASONS FOR CAUTION Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-. WIDER IMPLICATIONS OF THE FINDINGS Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics. STUDY FUNDING AND COMPETING INTEREST(S) The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

Exposure to ambient fine particulate matter and semen quality in Taiwan

Abstract: Objectives Environmental exposure to chemicals has been considered a potential factor contributing to deteriorated semen quality. However, previous literature on exposure to air pollution and semen quality is inconsistent. We therefore investigated the health effects of short-term and long-term exposure to fine particulate matter (PM 2.5 ) on semen quality in Taiwanese men from the general population. Methods A cross-sectional study was conducted among 6475 male participants aged 15–49 years who participated in a standard medical examination programme in Taiwan between 2001 and 2014. Semen quality was assessed according to the WHO 1999 guidelines, including sperm concentration, total motility, progressive motility and morphology. Three-month and 2-year average PM 2.5 concentrations were estimated at each participant’s address using a spatiotemporal model based on satellite-derived aerosol optical depth data. Multivariable linear and logistic regressions were used to examine the associations between PM 2.5 and semen quality. Results A robust association was observed between exposure to PM 2.5 and decreased normal morphology. Every increment of 5 µg/m 3 in 2-year average PM 2.5 was significantly associated with a decrease of 1.29% in sperm normal morphology and a 26% increased risk of having the bottom 10% of sperm normal morphology, after adjusting for a wide range of potential confounders (p 3 in 2-year average PM 2.5 was associated with an increase of 1.03×10 6 /mL in sperm concentration and a 10% decreased risk of being the bottom 10% of sperm concentration (both p 2.5 . Conclusions Exposure to ambient PM 2.5 air pollution is associated with a lower level of sperm normal morphology and a higher level of sperm concentration.

Quality of semen and histological analysis of testes in Eurasian perch Perca fluviatilis L. during a spawning period

Abstract: A qualitative assessment of the Eurasian perch Perca fluviatilis semen describing the basic parameters of seminal plasma was completed. The histological methods used in this study showed changes in perch gonads during a spawning and post-spawning period. At the late period of the spawning season, the structure of testes was clearly loosened and spermatozoa did not fill uniformly all the ampullae of the testes, leaving free spaces at their banks and no spermatids were observed. The results confirmed that there was an additional period after spawn - ing in the annual reproductive cycle of the male Eurasian perch. In both years of investigations (2000-2001), an essential decline in sperm motility at the late period of the spawning season was observed, from more than 85% to 56% in 2000. The sperm motility was not influenced by sperm concentrations because throughout the spawn - ing time no changes in the sperm concentration were observed, 32.4 and 32.6 mld/ml at beginning and at the late period of spawning period, respectively. In contrast to sperm concentrations, protein concentrations in seminal plasma increased in the late period of spawning season, from 3.95 to 5.16 g/l in 2001. This study confirmed that protein concentrations in the seminal plasma of most fish are much lower than in the other vertebrates. Among the fish examined, perch is characterized by one of the highest values of protein concentrations in plasma. Our observations confirmed that a high water temperature influenced anatomical and functional parameters of the reproductive system in male perch.

The Effect of Glyphosate on Human Sperm Motility and Sperm DNA Fragmentation.

Abstract: Glyphosate is the active ingredient of Roundup®, which is one of the most popular herbicides worldwide. Although many studies have focused on the reproductive toxicity of glyphosate or glyphosate-based herbicides, the majority of them have concluded that the effect of the specific herbicide is negligible, while only a few studies indicate the male reproductive toxicity of glyphosate alone. The aim of the present study was to investigate the effect of 0.36 mg/L glyphosate on sperm motility and sperm DNA fragmentation (SDF). Thirty healthy men volunteered to undergo semen analysis for the purpose of the study. Sperm motility was calculated according to WHO 2010 guidelines at collection time (zero time) and 1 h post-treatment with glyphosate. Sperm DNA fragmentation was evaluated with Halosperm® G2 kit for both the control and glyphosate-treated sperm samples. Sperm progressive motility of glyphosate-treated samples was significantly reduced after 1 h post-treatment in comparison to the respective controls, in contrast to the SDF of glyphosate-treated samples, which was comparable to the respective controls. Conclusively, under these in vitro conditions, at high concentrations that greatly exceed environmental exposures, glyphosate exerts toxic effects on sperm progressive motility but not on sperm DNA integrity, meaning that the toxic effect is limited only to motility, at least in the first hour.

Obesity impairs male fertility through long-term effects on spermatogenesis

Abstract: This study aimed to investigate the effect and possible underlying mechanisms of high-fat diet-induced obesity on spermatogenesis in male rats. A total of 45 male rats were randomly divided into control (n = 15, normal diet) and obesity groups (n = 30, high-fat diet) and were fed for 16 weeks. Body weight and organ indexes were determined after sacrifice. Indicators of reproductive function, including sperm count, sperm motility, apoptosis of spermatogenic cells, and oxidative stress levels, were measured. Serum metabolic parameters and reproductive hormones were also assayed. Compared with the control group, epididymal sperm motility in the obese rats was significantly decreased (P 0.05). Nutritional obesity can damage spermatogenesis in male rats due to long-term effects on spermatogenesis.

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay

Abstract: Study question Can a discriminant threshold be determined for human sperm DNA oxidation? Summary answer A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. What is known already Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa; optimized consensus protocols are lacking and no thresholds of normality have been established. Study design, size, duration Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. Participants/materials, setting, methods This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). Main results and the role of chance Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. Limitations, reasons for caution The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. Wider implications of the findings Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. Study funding/competing interest(s) This work was funded by institutional grants from the CNRS, INSERM and Universite Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

Sperm motility in fish: technical applications and perspectives through CASA-Mot systems

Abstract: Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully.

Protective effect of L-carnitine and L-arginine against busulfan-induced oligospermia in adult rat.

Abstract: Summary Busulfan is an anticancer drug caused variety of adverse effects for patients with cancer. But it could cause damage to the male reproductive system as one of its adverse effects. This study aimed to investigate the protective effect of L-carnitine and L-arginine on semen quality, oxidative stress parameters and testes cell energy after busulfan treatment. Adult male rats were divided into four groups: control (Con), busulfan (Bus), busulfan plus L-arginine (Bus + L-arg) and busulfan plus L-carnitine (Bus + L-car). After 28 days, the semen was collected from the epididymis and the testes were assessed. Sperm count, motility and velocity were measured by CASA, and smears were prepared for assessment of sperm morphology. Serum and testes supernatants were separated for DNA metabolites, oxidative stress and cell energy parameters. Testes tissues also subjected for caspase-3. The results showed significant improvement in sperm morphology, motility, velocity and count in the groups treated with L-arginine and L-carnitine and accompanied with an increase in MDA, GSSG and ATP, reduction in GSH, AMP, ADP, NO and 8-OHDG also recorded. These results are supported by caspase-3. Conclusions: Administration of L-arg and L-car attenuated the cytotoxic effects of busulfan by improving semen parameters, reducing oxidative stress and maintaining cell energy.

Comparative analysis of the protective effects of curcumin and N -acetyl cysteine against paracetamol-induced hepatic, renal, and testicular toxicity in Wistar rats

Abstract: This study aimed to investigate the possible protective role of curcumin (CUR) vs. N-acetyl cysteine (NAC) against paracetamol (PCM)-induced oxidative damage and impairment of liver, kidney, and testicular functions, as well as hematotoxicity, in albino rats. A large single dose of PCM induced lipid peroxidation along with a significant decline in glutathione content and catalase activity in the liver, kidneys, and testicles. The apparent oxidative damage was associated with evident hepatic, renal, and testicular dysfunction, which was confirmed in histopathological lesions, and increased serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. PCM decreased serum total protein, albumin, and globulin contents; increased bilirubin, urea, and creatinine contents; and induced hematotoxicity. PCM also reduced the sperm cell count, sperm motility, and alive sperm rate and increased the sperm abnormality rate. Pretreatment of PCM-intoxicated animals with CUR or NAC substantially alleviated the increase in malondialdehyde and maintained the antioxidants at control levels. These pretreatments also minimized liver, kidney, and testicular histopathological changes and normalized their functions. CUR similarly mitigated the PCM hemato- and hepatotoxicity compared with NAC. However, it exhibited a pronounced nephroprotection, rather than reproductive protection as did NAC. Our findings demonstrate that a large single dose of PCM is not only associated with hepatotoxicity but also nephrotoxicity and reproductive toxicity. Both CUR and NAC administration provided substantial organ protection with pronounced efficacy against PCM nephrotoxicity with CUR and reproductive toxicity with NAC, which was possibly mediated through their antioxidant activities, as well as their specific characteristics.

Proteomic profile of human spermatozoa in healthy and asthenozoospermic individuals.

Abstract: Asthenozoospermia is considered as a common cause of male infertility and characterized by reduced sperm motility. However, the molecular mechanism that impairs sperm motility remains unknown in most cases. In the present review, we briefly reviewed the proteome of spermatozoa and seminal plasma in asthenozoospermia and considered post-translational modifications in spermatozoa of asthenozoospermia. The reduction of sperm motility in asthenozoospermic patients had been attributed to factors, for instance, energy metabolism dysfunction or structural defects in the sperm-tail protein components and the differential proteins potentially involved in sperm motility such as COX6B, ODF, TUBB2B were described. Comparative proteomic analysis open a window to discover the potential pathogenic mechanisms of asthenozoospermia and the biomarkers with clinical significance.

Cerium oxide nanoparticle elicits oxidative stress, endocrine imbalance and lowers sperm characteristics in testes of balb/c mice

Abstract: The toxicity of metallic nanoparticles is a growing concern due to its application in industries and homes. We investigated the toxicity of cerium oxide nanoparticles (CeO2 NPs) on reproductive system in male balb/c mice. Twenty mice were divided into four groups of five animals each and treated thus: normal saline (control), 100, 200 and 300 μg/kg CeO2 NPs (i.p.,) thrice in a week for five consecutive weeks. Results showed that CeO2 NPs significantly reduced the levels of haemoglobin, PCV and RBC count relative to controls. In addition, luteinising and follicle-stimulating hormones (FSH and LH) and prolactin were significantly reduced in the mice. Specifically, CeO2 NPs at 100 μg/kg decreased testosterone by 23%, while CeO2 NPs at 200 μg/kg decreased FSH, LH and prolactin by 25%, 26% and 13%, respectively. Testicular malondialdehyde was increased by 103%, 106% and 135% in mice treated with 100, 200 and 300 μg/kg CeO2 NPs, respectively. CeO2 NPs caused a significant reduction in activities of antioxidant enzymes and levels of reduced glutathione and total nitric oxide. Moreso, CeO2 NPs decreased sperm motility and count and increased total sperm abnormality in mice. Histology revealed congestion and degeneration of seminiferous tubules. Overall, CeO2 NPs induces testicular dysfunction via disruption of antioxidant/oxidant balance and endocrine suppression.

Abnormal sperm concentration and motility as well as advanced paternal age compromise early embryonic development but not pregnancy outcomes: a retrospective study of 1266 ICSI cycles.

Abstract: To investigate the effect of sperm concentration, motility and advanced paternal age on reproductive outcomes. A retrospective analysis of 1266 intracytoplasmic sperm injection (ICSI) cycles between 2013 and 2017. The cohort was divided into four groups according to semen concentration based on the WHO criteria (2010): group A (conc. <1 M/ml), group B (1 ≤ conc. <5 M/ml), group C (5 ≤ conc. < 15 M/ml) and the control group D (conc. ≥15 M/ml). The primary outcome investigated was the blastulation rate. Secondary outcomes were fertilization rate, top quality blastocyst formation rate and ongoing pregnancy rate. After adjustment for maternal age and number of oocytes recovered, a significant difference was observed between group A and group D on the rate of fertilized oocytes [66.7 (40.0–80.0) vs 75.0 (57.1–90.2), adjusted p < 0.001] and the blastocyst formation rate [50.0 (33.3–66.3) vs 55.6 (40.0–75.0), adjusted p < 0.05]. However, the male factor did not affect the top quality blastocyst formation rate nor the ongoing pregnancy rate. Considering the age of the male partner as confounding factor, at the increase of each year of age, a reduction of 0.3% on the fertilization rate was observed but no other outcome was impacted. A negative correlation was also observed between sperm motility and fertilization rate in the group with a motility <5%. Male factor infertility and advanced paternal age may compromise fertilization and blastulation rates but not top quality blastocyst formation rate or the establishment of pregnancy in ICSI cycles.

Parabens generate reactive oxygen species in human spermatozoa.

Abstract: Parabens are used as antimicrobial preservative agent in many commercial products including cosmetics and pharmaceuticals. Weak oestrogenic and antiandrogenic activities have been attributed to parabens in in vitro and in vivo studies. In this study, human spermatozoa were exposed to different concentrations of an equimolar paraben mixture containing methyl, ethyl, propyl and butylparaben as well as to methylparaben alone at a concentration that is typical of commercially available vaginal lubricants. The induction of oxidative stress and DNA damage was then assessed at different time points. Our results demonstrate that the paraben mixture was capable of stimulating the generation of mitochondrial and cytosolic reactive oxygen species (ROS), inhibiting sperm motility and viability in a dose-dependent manner. The ability of individual parabens to activate ROS generation and induce oxidative DNA damage was related to alkyl chain length. At the concentration used clinically, methylparaben inhibited sperm motility after both 2 and 5 h exposure (p < 0.05) and affected cell viability (p < 0.01) while augmenting ROS production and oxidative DNA damage. However, DNA fragmentation was not evident following methylparaben exposure. Based on these results, we conclude that, at the concentrations used in commercially available formulations, parabens may impair sperm motility, enhance the generation of mitochondrial ROS and stimulate the formation of oxidative DNA adducts. Taken together, these data underline the potential cytotoxic and genotoxic impact of such compounds in a clinical setting.

Relationships between seminal plasma composition and sperm quality parameters of the Salmo trutta macrostigma (Dumeril, 1858) semen: with emphasis on sperm motility

Abstract: The mineral and organic composition of seminal plasma, physical spermatological parameters and their physiological relationships were investigated in Salmo trutta macrostigma. The seminal plasma