Motile Human Spermatozoa Research Articles

P–081 Microfluidic sorting does not improve clinical outcomes compared to magnetic activated cell sorting (MACS) in Assisted Reproduction

Abstract Study question Does microfluidic sorting improve clinical outcomes over magnetic activated cell sorting (MACS) in ovum donation cycles? Summary answer Performing microfluidic sorting does not seem to improve clinical outcomes compared to MACS in ovum donation cycles. What is known already Novel sperm selection techniques, such as magnetic activated cell sorting (MACS), have been described as useful procedures to increase reproductive outcomes when male factor is present. Because of centrifugation steps associated to swim-up or density-gradient can induce sperm DNA fragmentation, microfluidic sperm sorters are being used to isolate motile human spermatozoa based on fluid dynamics and avoiding sample manipulation. This new technology has been shown to reduce the level of sperm DNA damage, especially double strand breaks, but an improvement of clinical outcome by using this technique remain unclear. Study design, size, duration Prospective and observational study to evaluate the efficacy of a sperm sorting technique based on microfluidic technology versus a technique based on the removal of apoptotic spermatozoa by MACS. The study was performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. All men attending for an ovum donation cycle during the aforementioned study period were included. The exclusion criteria were sperm concentration <5 mill/mL and <15% of progressive motile spermatozoa. Participants/materials, setting, methods Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. Subsequently, each of the processed sperm samples was used to microinject half of the oocytes obtained during oocyte retrieval. In all case, a single-embryo transfer was performed. Variables were expressed as mean values and standard deviations. Statistical analysis was performed by ANOVA and Chi-squared where applicable; significance established under 0.05. Main results and the role of chance We included 48 couples in the study; of these, n = 31 transferred an embryo derived from a MACS processed sperm sample, while n = 17 received an embryo from the microfluidic one. Groups were homogeneous in terms of number of transferred embryos and frozen embryos, usable blastocyst rate and fertilization rate; results were as follows for MACS and microfluidic processing respectively: number of transferred embryos (1.0±0.0 vs. 1.0±0.0, p = 0.978); number of frozen embryos (1.6±0.5 vs. 1.4±0.7 p = 0.168); usable blastocyst rate (40.7% vs. 43.1%, p = 0.384); and fertilization rate (80.4% vs. 75.3%, p = 0.075). However, according clinical outcomes, we observed significant differences in implantation rate (74.2% vs. 58.8%, p < 0.001) and in clinical pregnancy rate (74.2% vs. 58.8%, p < 0.001); finally, the miscarriage rate was similar between the two groups of study (6.4% vs. 5.8%, p = 0.876). Limitations, reasons for caution This study has not considered the indication in male factor couples due to a high degree of double-strand DNA fragmentation. Therefore, more specific studies are required to determine in which patients, microfluidics sorter selection would significantly improve clinical outcomes. Wider implications of the findings: In an unselected population, magnetic activated cell sorting significantly improves clinical outcomes compared to a microfluidic technique, so this latter method should not be recommended without a male factor indication associated with sperm DNA damage. The proposed microfluidic technology does not seem to offer a flow-free approach to select spermatozoa. Trial registration number NCT04061486

Spiral Trajectory Modulation of Rheotaxic Motile Human Sperm in Cylindrical Microfluidic Channels of Different Inner Diameters.

We investigated the relationship between human sperm rheotaxis and motile sperm trajectories by using poly-(dimethylsiloxane) (PDMS)-based cylindrical microfluidic channels with inner diameters of 100 μm, 50 μm, and 70 μm, which corresponded to the inner diameter of the human isthmus, the length of a sperm and a diameter intermediate between the two, respectively. We counted the number of rheotaxic sperm and sperm with spiral motion. We also analyzed motile sperm trajectories. As the cylindrical channel diameter was decreased, the percentage of sperm cells exhibiting rheotaxis, the percentage of sperm cells exhibiting spiral motion, the frequency-to-diameter ratio of the sperm cells' spiral trajectories, and the surface area of the microfluidic channel increased, while the flagellar motion at the channel wall decreased. The percentage of sperm exhibiting a spiral trajectory and the frequency-to-diameter ratio of the sperm cells' spiral trajectories were thus affected by the channel diameter. Our findings suggest that the oviduct structure affects the swimming properties of sperm cells, guiding them from the uterus to the ampulla for egg fertilization. These results could contribute to the development of motile sperm-sorting microfluidic devices for assisted reproductive technologies.

Evidence for Rapid Oxidative Phosphorylation and Lactate Fermentation in Motile Human Sperm by Hyperpolarized 13C Magnetic Resonance Spectroscopy

Poor sperm motility is a common cause of male infertility for which there are no empirical therapies. Sperm motility is powered by adenosine triphosphate but the relative importance of lactate fermentation and Oxidative Phosphorylation (OxPhos) is debated. To study the relationship between energy metabolism and sperm motility we used dissolution Dynamic Nuclear Polarization (dDNP) for the first time to show the rapid conversion of 13C1-pyruvate to lactate and bicarbonate, indicating active glycolytic and OxPhos metabolism in sperm. The magnitude of both lactate and bicarbonate signals were positively correlated with the concentration of progressively motile sperm. After controlling for sperm concentration, increased progressive sperm motility generated more pyruvate conversion to lactate and bicarbonate. The technique of dDNP allows ‘snapshots’ of sperm metabolism to be tracked over the different stages of their life. This may provide help to uncover the causes of poor sperm motility and suggest new approaches for novel treatments or therapies.

Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.

Potential targetability of multi-walled carbon nanotube loaded with silver nanoparticles photosynthesized from Ocimum tenuiflorum (tulsi extract) in fertility diagnosis

Nanocarrier mediated targeted delivery and biosensing in reproductive health care is a major exploratory domain. This work demonstrates the loading of silver nanoparticle (AgNP) inside the multiwalled carbon nanotube (MWCNT) and their targetability to the intracellular part of the sperm cell for its further application in biosensing based infertility diagnosis. Ocimum tenuiflorum (tulsi extract) mediated photosynthesized AgNP exhibited spherical shape, 5–40 nm size and surface plasmonic resonance at 430 nm. After loading of freshly prepared AgNP into emulsified MWCNT, the loading was confirmed with spectroscopic and microscopic methods. FTIR analysis displayed significant shifting at 3450 cm−1 (–OH stretching) and 1615 cm−1 (CNT back bone) which validated the binding of AgNP with MWCNT and interestingly heat flow analysis revealed that Ag loaded MWCNT has greater stability than AgNP. Moreover, AFM based surface profile height analysis clearly showed the loading of AgNP inside MWCNT as surface height of MWCNT increased from 22 to 32 nm, which in turn confirmed the encapsulation of 10 nm size of AgNP inside the tube. Furthermore, surface enhanced Raman spectroscopy (SERS) confirmed the homogeneous loading as there were no changes in D/G ratio. SERS analysis for the interaction of AgNP loaded MWCNT with freshly collected healthy, motile human spermatozoa showed a significant Raman shift at 800–780 cm−1 (NH2+ twist) and 1050–1060 cm−1 (vas PO3−) for change in DNA packaging process and its stabilizing protein polyamine respectively. Finally, DNA fragmentation and morphological examination confirmed the binding and targetability of AgNP to the sperm nucleus. Improved targeting efficiency and biosenssing ability make AgNP-MWCNT composite suitable in fertility diagnosis.

Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos

Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 107 spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 × 106 group (10 × 106 sperm/mL) were higher than in the 1 × 106 and 5 × 106 groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation.

Two-dimensional slither swimming of sperm within a micrometre of a surface.

Sperm motion near surfaces plays a crucial role in fertilization, but the nature of this motion has not been resolved. Using total internal reflection fluorescence microscopy, we selectively imaged motile human and bull sperm located within one micron of a surface, revealing a distinct two-dimensional (2D) ‘slither' swimming mode whereby the full cell length (50–80 μm) is confined within 1 μm of a surface. This behaviour is distinct from bulk and near-wall swimming modes where the flagellar wave is helical and the head continuously rotates. The slither mode is intermittent (∼1 s, ∼70 μm), and in human sperm, is observed only for viscosities over 20 mPa·s. Bull sperm are slower in this surface-confined swimming mode, owing to a decrease in their flagellar wave amplitude. In contrast, human sperm are ∼50% faster—suggesting a strategy that is well suited to the highly viscous and confined lumen within the human fallopian tube.

Paper-based diagnostic devices for evaluating the quality of human sperm

Male infertility, which amounts to half of all infertility cases, is a serious problem worldwide. The percentage of fertility-related patients in sub-Saharan African countries is higher than that for the developed countries. In low-resource countries, examination of sperm characteristics for male infertility cannot be undertaken because of poor clinical access. To evaluate male fertility in assisted reproductive medicine laboratories, the numbers of motile human sperm, the degree of sperm motility, and sperm morphology have been commonly analyzed using a microscope. It is challenging to monitor the health status of human sperm in resource-limited or remote settings for two primary reasons: (1) high capital cost (equipment for currently accepted procedural standard), and (2) complexity of the currently accepted procedural standard used to simultaneously measure human sperm concentration and motility by skillful embryologists. Determining the health status of human sperm in order to evaluate fertilization capacity using various types of low-cost, easy-to-use, and rapid devices (or systems) is a longstanding but interesting biotechnologically relevant issue in various scientific communities such as male reproduction. Furthering such efforts will inherently influence birth rate in both developed and developing nations. We have demonstrated an inexpensive but robust and easy-to-handle device for monitoring the health status of human sperm made by patterning a piece of paper and measuring the activity of a specific enzyme—a simple and elegant solution. After applying semen to the hydrophilic center circle of our patterned paper, a thiazine assay can be used to suggest sperm concentration in semen, and a tetrazolium-based colorimetric assay (MTT assay) data can be used to help estimate the percentage of motile human sperm (sperm motility) in semen based on the character that motile human sperm moved in and on the paper. Using this paper-based device, we can evaluate fertility levels without consulting doctors and use our assay to compare results with World Health Organization (WHO) reference values for sperm concentration (>2×107) and motility (>50 %). The duration and cost of one entire test are 30 min and 0.1 USD, respectively. We believe that this paper-based assay system would be useful for fertility checks based on WHO references, without need of a microscope, at home. Using this assay method, males in developed or developing countries who are reluctant or unable to consult assisted reproductive technologies clinics can self-analyze their sperm characteristics. We further note that our approach adheres to WHO regulations, especially in regard to in vitro diagnostic device performance with an associated diagnostic algorithm to enhance diagnostic accuracy (compared with just one diagnostic output), and we wish to emphasize that our research could significantly advance a broad range of diagnostic developments including paper-based diagnostic devices, in vitro diagnostic devices, and diagnosis of other diseases in various divisions of translational medicine. These results, we believe, will be of interest to a wide scientific audience working in materials science (biomaterials), chemistry (analytical and clinical), lab-on-a-chip technologies (the development of diagnostic tools), reproductive medicine, bioengineering, and translational medicine.

Co-Incubation of Human Spermatozoa with Anti-VDAC Antibody Reduced Sperm Motility

Background: Voltage-dependent anion channel (VDAC), a channel protein, exists in the outer mitochondrial membrane of somatic cells and is involved in multiple physiological and pathophysiological processes. Up until now, little has been known about VDAC in male germ cells. In the present study, the relationship between VDAC and human sperm motility was explored. Methods: Highly motile human spermatozoa were incubated in vitro with anti-VDAC antibody. Total sperm motility, straight line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP) were recorded. Intracellular free calcium concentration ([Ca²⁺]_i), pH value (pHi), and ATP content were determined. Results: Co-incubation with anti-VDAC antibody reduced VSL, VCL, and VAP of spermatozoa. Co-incubation further reduced [Ca²⁺]_i. Anti-VDAC antibody did not significantly alter total sperm motility, pHi and intracellular ATP content. Conclusion: The data suggest that co-incubation with anti-VDAC antibody reduces sperm motility through inhibition of Ca²⁺ transmembrane flow. In this way, VDAC participates in the modulation of human sperm motility through mediating Ca²⁺ transmembrane transport and exchange.

Motile Human Sperm Sorting by an Integrated Microfluidic System

The integrated microfluidic system consists of laminar stream-based microchannels and flow cytometric analysis to enhance the human sperm motility sorting efficiency. This modified sperm sorter provided a new selection that had advances in resolving traditional sorting problems. In order to design the sorting sorting device, CFDRC simulation was performed to analyze the phenomenon of 3-D flow field. Microscopic examination revealed that sperms with different motility. For the purpose of confirming the real sorting situation, the proportions of living and dead sperm after sorting were readily identified and quantified through use of flow cytometric analysis. The viability results of sample injected from two-outlet and four-outlet chip were 95.2% and 92.2% that presented the higher quality of sorting sperm. As a result, more amounts of living sperms that mean motile sperms were counted in four-outlet channels. With this approach, the characteristics of laminar flow and a novel design were utilized to demonstrate the microfluidic system with high efficiency for sperm motility sorting. Therefore, the optimized microfluidic system provides a good opportunity to use and an inexpensive requirement to select the most appropriate sperms before IVF (in vitro fertilization) process.

Evaluation of the spermicidal and contraceptive activity of Platycodin D, a Saponin from Platycodon grandiflorum.

BackgroundThe extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity, as well as the safety, of Platycodin D (PD), a major saponin in Platycodon grandiflorum.MethodsUsing the computer-aided sperm analysis (CASA) test criteria, the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation.ResultsA dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD, indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats, was non-DNA damaging and was not harmful to the vaginal tissue in the rats.ConclusionPD has significant spermicidal activity that should be explored in further studies.

001 Milestones in cryobiology

Cryobiology is a discipline that has its origins in experiments that were published exactly 350 years ago. In 1663, Henry Power, a British physician, reported that he had used a mixture of snow and salt to freeze nematodes. When he thawed the samples three hours later, he stated that “all my little Animals made their re-appearance and danced and frisked about as lively as ever.” Over the centuries, this discipline has evolved and grown into a formal science that ranges from theoretical analysis of the behavior of solutions and the response of biological cells at subzero temperatures to application of very practical procedures to cryopreserve 250 million doses of cattle sperm annually as well as sperm of more than 60 species of mammals, fish and birds. Cryobiology now plays a critically important role in human and animal medicine, agriculture, research and as a fundamental discipline itself. Its applications are so diverse that it is not possible to even mention all of the milestones that have been achieved. Throughout the 18th century, scientists studied the phenomenon of cryptobiosis, the interruption of normal metabolism of cells and simple organisms by cooling them to low temperatures. By the 19th century, such studies had developed into formal experimental biology in which systematic measurements of the effects of freezing were made. By the 1920s and 30s, investigators were attempting to preserve cells of many animal species by cooling them to temperatures as low as that of liquid nitrogen and even liquid helium. In some isolated cases, cells of various types survived; for example, Jahnel in Germany observed motile human spermatozoa that had been cooled to −269 °C. In the 1940s and 1950s, several observations significant enough to be called milestones had been made. These included the demonstration by Polge and his colleagues that glycerol protected rooster spermatozoa against the damaging effects of freezing. Within a short time, this finding had been extended to cattle and human spermatozoa and then to erythrocytes. Freezing of sperm resulted in live births of calves and children. To search for possible causes of injury, Luyet and Meryman independently studied formation and re-crystallization of ice crystals in aqueous solutions. Lovelock began to apply formal physical–chemical analysis to the subject. Other biologists measured effects of freezing and thawing on a wide variety of microorganisms and animal cells. In the 1950s, there was sufficient interest in these observations of freezing to justify convening several symposia. In the 1960s, Mazur formulated his mathematical analysis of the response of cells when they are frozen. Shortly thereafter, it was demonstrated that cells of higher animals subjected to freezing and thawing exhibited responses very similar to those of microorganisms. This, in turn, led to the successful cryopreservation of numerous types of animal and plant cells. In the 1970s, application of fundamental principles resulted in the successful cryopreservation of mouse embryos and then to those of more than 25 other species. Then, procedures were devised to cryopreserve organized tissues of many types and from diverse species. Now, in the 21st century, an incredible variety of cells and tissues of plants and animals are routinely preserved not only by freezing but also by vitrification and even by drying. Many of these achievements were first attempted by the earliest investigators of cryptobiosis. Source of funding: None declared. Conflict of interest: None declared. sleibo@uno.edu

The three-dimensional image analysis of the chromocenter in motile and immotile human sperm

Chromosomes in human spermatozoa are arranged non-randomly with the centromeres of non-homologous chromosomes forming a chromocenter. We have compared motile and immotile sperm populations in normozoospermic patients to determine if there is any dissimilarity in the formation of the chromocenter and the nuclear position of chromosome 17. Based on the differences between motile and immotile populations, we propose for the ‘optimal’ nuclear organization to be defined as containing 1 to 3 chromocenter(s) with central radial and median longitudinal position for the centromere of chromosome 17. By this definition, 42% of motile spermatozoa had ‘optima’ nuclei, in comparison to 25% of immotile spermatozoa (P < 0.05). Immotile spermatozoa exhibited a greater disruption in the formation of the chromocenter, altered position of the centromere of chromosome 17, and were more prone to chemical decondensation, resulting in higher nuclear and chromocenter volumes. The altered topology of the chromosomes might lead to the disruption of the sequence of events involved in fertilization and early embryonic development.

Cryopreservation of Human Spermatozoa Decreases the Number of Motile Normal Spermatozoa, Induces Nuclear Vacuolization and Chromatin Decondensation

Even though cryopreservation of human spermatozoa is known to alter sperm motility and viability, it may also induce nuclear damages. The present study set out to determine whether or not cryopreservation alters motile sperm morphology under high magnification and/or is associated with chromatin decondensation. For 25 infertile men, we used high-magnification microscopy to determine the proportions of various types of motile spermatozoa before and after freezing-thawing: morphometrically normal spermatozoa with no vacuole (grade I), ≤ 2 small vacuoles (grade II), at least 1 large vacuole or >2 small vacuoles (grade III), and morphometrically abnormal spermatozoa (grade IV). The spermatozoa's chromatin condensation and viability were also assessed before and after freezing-thawing. Cryopreservation induced sperm nuclear vacuolization. It decreased the proportion of grade I + II spermatozoa (P < .001). It induced a decrease in the sperm viability rate (P < .001) and increased the proportion of sperm with noncondensed chromatin (P < .001). The latter parameter was strongly correlated with sperm viability (r = 0.71; P < .001). However, even motile sperm presented a failure of chromatin condensation after freezing-thawing, because the proportion of sperm with noncondensed chromatin was correlated with high-magnification morphology (r = -0.49 and 0.49 for the proportions of grade I + II and grades III + IV, respectively; P < .001). Cryopreservation alters the organelle morphology of motile human spermatozoa and induces sperm chromatin decondensation. High-magnification microscopy may be useful for evaluating frozen-thawed spermatozoa before use in assisted reproductive technology procedures (such as intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection) and for performing research on cryopreservation methods. If frozen-thawed sperm is to be used for intracytoplasmic sperm injection, morphological selection under high magnification may be of particular value.

Potassium channel in human sperm identified by whole cell patch clamping plays a role in normal sperm physiology

To characterise and identify the functional role of potassium channels in motile human sperm by whole cell patch clamping. Whole cell patch clamping of motile human sperm was used to identify a native K+ conductance. Pharmacology and ionic substitution characterised the biophysical properties of the channel. Function assays in the presence and absence of identified channel blockers were used to identify of the role of the channel in normal sperm function. Spermatozoa from healthy donors with normal semen parameters (WHO 2010) were isolated by swim up and incubated under capacitating conditions for 4 hours (25mM NaHCO3, 6% CO2 at 37°C). Currents were recorded under voltage clamp using ramp and leak subtracted step protocols (-80 to 80mV, holding potential -80mV). Standard pipette and bath solutions were designed to mimic quasi-physiological ionic conditions. Motility was assessed using computer aided sperm analysis and Kremer tests were used to assess the cells ability to penetrate viscous media in the presence of the putative potassium channel blocker Quinidine. Chi2, ANOVA and students t-test were used to identify statistical significance where appropriate. Statistical significance P<0.05. Data showed an outwardly rectifying current only active when depolarised past -40mV. Replacing pipette potassium with cesium (n=8) abolished the depolarization-induced current. Putative K+ blocker quinidine [0.3mM] completely inhibited the outward current, depolarised the resting membrane potential, reduced progressive motility (n=9) and penetration into viscous media (n=6) by ∼50% and 70% respectively (P<0.001). Qunidine also reduced straight line velocity and average path velocity by 22 and 31%, respectively (P<0.005). Human sperm possess K+ channels involved in normal sperm function that may play a role in male fertility. This is the first time this has been documented in mature human sperm.

Relationship of DNA integrity of motile human spermatozoa and ICSI outcome

Relationship of DNA integrity of motile human spermatozoa and ICSI outcome

Human spermatozoa migration in microchannels reveals boundary-following navigation

The migratory abilities of motile human spermatozoa in vivo are essential for natural fertility, but it remains a mystery what properties distinguish the tens of cells which find an egg from the millions of cells ejaculated. To reach the site of fertilization, sperm must traverse narrow and convoluted channels, filled with viscous fluids. To elucidate individual and group behaviors that may occur in the complex three-dimensional female tract environment, we examine the behavior of migrating sperm in assorted microchannel geometries. Cells rarely swim in the central part of the channel cross-section, instead traveling along the intersection of the channel walls ("channel corners"). When the channel turns sharply, cells leave the corner, continuing ahead until hitting the opposite wall of the channel, with a distribution of departure angles, the latter being modulated by fluid viscosity. If the channel bend is smooth, cells depart from the inner wall when the curvature radius is less than a threshold value close to 150μm. Specific wall shapes are able to preferentially direct motile cells. As a consequence of swimming along the corners, the domain occupied by cells becomes essentially one-dimensional, leading to frequent collisions, and needs to be accounted for when modeling the behavior of populations of migratory cells and considering how sperm populate and navigate the female tract. The combined effect of viscosity and three-dimensional architecture should be accounted for in future in vitro studies of sperm chemoattraction.

Role of the Serine/Threonine Phosphatases During the Progesterone-Induced Acrosome Reaction and Increase in Intracellular Free Calcium Concentration in Human Sperm.

The acrosome reaction (AR) is an exocytotic event that takes place by fusion of the plasma membrane and the outer acrosomal membrane in sperm previously capacitated. It is widely accepted that progesterone stimulates human sperm AR and that acrosomal exocytosis is accompanied by a transient elevation of intracellular free Ca2+ concentration ([Ca2+]I), which is a consequence of a Ca2+ influx. It is known that during capacitation changes occur in the phosphorylation state of proteins, mediated by protein kinases (PKs) and phosphatase (PPs). However, it is not yet clear whether something similar takes place during the AR. The aim of this work was to study the role of serine/threonine PPs during the progesterone-induced AR and increase of [Ca2+]L, using specific inhibitors. Motile human sperm, free of seminal plasma, were obtained with a Percoll gradient and then incubated in Tyrode medium (2.6% BSA, 37 °C, 5% CO2). After 18 hr, aliquots were treated with different inhibitors of PPs by 15 min. The inhibitors used were 0.1 nM deltamethrin, 10 nM FK-506, and 10 nM cyclosporine A for PP2B; 90 nM endothal and 0.1 nM okadaic acid for PP2A; and 10 nM okadaic acid for PP1. Then, 7 µM progesterone was added and incubated for extra 15 minutes. Acrosomal status and viability were evaluated using PSA-FITC and Hoechst 33258, respectively. To assess the effect of the PPs inhibitors on [Ca2+]I, capacitated sperm were incubated for 30 minutes with 3 µM fura 2AM; after 15 min inhibitors were added. Calcium influx was stimulated with 7 µM progesterone. The fluorescence was monitored with a spectrofluorometer at an excitation wavelength pair of 340/380 nm and emission wavelength of 510 nm. The results indicated that most PPs inhibitors do not affect the induction of the AR by progesterone or the increase in [Ca2+]I. However, two inhibitors of PP2B, FK506 and cyclosporine A, inhibited the progesterone-induced AR but they did not have an effect on the increase of [Ca2+]I. These results suggest that PP2B (calcineurin) may be involved in the exocytosis of the acrosome induced by progesterone but not in the Ca2+ influx that precedes it. The mechanism of this inhibition is still unknown. Fondecyt 1080028. (poster)

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 μm² vs. 18.8 ± 1.4 μm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Mobile Phone Radiation Does Not Induce Pro-apoptosis Effects in Human Spermatozoa

Abstract Recent reports suggest that mobile phone radiation may diminish male fertility. However, the effects of this radiation on human spermatozoa are largely unknown. The present study examined effects of the radiation on induction of apoptosis-related properties in human spermatozoa. Ejaculated, density-purified, highly motile human spermatozoa were exposed to mobile phone radiation at specific absorption rates (SARs) of 2.0 and 5.7 W/kg. At various times after exposure, flow cytometry was used to examine caspase 3 activity, externalization of phosphatidylserine (PS), induction of DNA strand breaks, and generation of reactive oxygen species. Mobile phone radiation had no statistically significant effect on any of the parameters studied. This suggests that the impairment of fertility reported in some studies was not caused by the induction of apoptosis in spermatozoa.