Percentage Of Hyperactivated Spermatozoa Research Articles

Impact of old age and season on Chios ram semen quality

The present study aimed to provide new and objective information regarding the effect of season and age on the characteristics of Chios ram semen. The semen collection was performed biweekly throughout a year, including both breeding and non-breeding periods. Seven active Chios rams were divided in two age groups: three rams of 2–6 years old (mature) and four rams of 9–13 years old (old). After collection, semen volume and sperm concentration were measured. One hundred sixty-eight (168) semen samples were collected, extended and evaluated. All samples were tested for motility and kinetics (Computer Assisted Sperm Analyzer), viability-membrane integrity (Eosin-Nigrosin), morphology (SpermBlue®), chromatin integrity (Acridine Orange Test) and sperm membrane biochemical functionality (Hypo-osmotic swelling test, HOST). Motility did not differ significantly between periods (p > 0.05) or between age groups (p > 0.05). The percentage of hyperactivated spermatozoa and head abnormalities was higher in the non-breeding compared to the breeding period (p = 0.011 and p = 0.001, respectively). Tail abnormalities were higher in the breeding (p = 0.022) and HOST + spermatozoa were lower in the non-breeding period (p < 0.001). Significantly higher values of semen volume (p = 0.001) and concentration (p < 0.001) were noticed in old compared to mature rams. In conclusion, old rams, up to the age of 13 years, preserve high semen quality, while season affects ram sperm membrane biochemical functionality and head morphology.

Effects of a micronutrient supplementation combined with a phosphodiesterase type 5 inhibitor on sperm quantitative and qualitative parameters, percentage of mature spermatozoa and sperm capacity to undergo hyperactivation: A randomised controlled trial.

The main objective of this study was to evaluate the effects of a micronutrient supplementation (MS) combined with avanafil on sperm function. Oligoasthenospermic men (n=217) were treated daily for 90days with either an MS (45 men, Group A), l-carnitine (44 men, Group B), MS plus avanafil (43 men, Group C) or avanafil (43 men, Group D); another group of 42 men with oligoasthenospermia (Group E) received no treatment. Sperm parameters were evaluated before and after the end of treatment in each Group A, B, C and D respectively. The same sperm parameters were measured in each participant of Group E before and at the 90-day experimental period. Within Groups A, C or D, the total percentage of motile spermatozoa, the hypoosmotic swelling test (HOST) result and the percentage of hyperactivated spermatozoa after incubation under conditions known to induce sperm capacitation were significantly greater after MS or MS plus avanafil treatment, or avanafil treatment than before the respective treatment. We suggest that MS or MS plus avanafil combined administration or avanafil alone improves sperm membrane permeability with an overall result improvement in sperm motility, outcome of HOST and increase in the percentage of hyperactivated spermatozoa.

Behavioral mechanism of human sperm in thermotaxis: a role for hyperactivation

What is the behavioral mechanism underlying the response of human spermatozoa to a temperature gradient in thermotaxis? Human spermatozoa swim up a temperature gradient by modulating their speed and frequencies of hyperactivation events and turns. Capacitated human spermatozoa are capable of thermotactically responding to a temperature gradient with an outcome of swimming up the gradient. This response occurs even when the gradient is very shallow. Human sperm samples were exposed to a fast temperature change. A quantitative analysis of sperm motility parameters, flagellar wave propagation, and directional changes before, during, and after the temperature change was carried out. The swimming behavior of 44 human sperm samples from nine healthy donors was recorded under a phase-contrast microscope at 75 and 2000 frames/s. A temperature shift was achieved by using a thermoregulated microscope stage. The tracks made by the cells were analyzed by a homemade computerized motion analysis system and ImageJ software. A temperature shift from 31 to 37°C resulted in enhanced speed and a lower frequency of turning events. These were reflected in a 35 ± 1% (mean ± SEM) increase of the straight-line velocity, 33 ± 1% increase of the average path velocity, 11 ± 1% increase of the curvilinear velocity, 20 ± 1% increase of the wobble, and 4 ± 1% increase of the linearity. Qualitatively, the inverse trend was observed in response to a 37-to-31°C shift. In addition, the amplitude of flagellar waves increased close to the sperm head, resulting in higher side-to-side motion of the head and, often, hyperactivation. This increase in the extent of sperm hyperactivation was reflected in an increase in the average (mean ± SEM) fractal dimension from 1.15 ± 0.01 to 1.29 ± 0.01 and in the percentage of hyperactivated spermatozoa from 3 ± 1% to 19 ± 2%. These changes in hyperactivation were observed less often in sperm populations that had not been incubated for capacitation. All these changes partially adapted within 3-10 min, meaning that following the initial change and while being kept at the new temperature, the values of the measured motility parameters slowly and partially returned toward the original values. These results led us to conclude that spermatozoa direct their swimming in a temperature gradient by modulating the frequency of turns (both abrupt turns as in hyperactivation events and subtle turns) and speed in a way that favors swimming in the direction of the gradient. The conclusions were made on the basis of results obtained in temporal and steep temperature gradients. The conclusions for spatial, shallow gradients were made by extrapolation. This is the first study that reveals the behavior of human spermatozoa in thermotaxis. This behavior is very similar to that observed during human sperm chemotaxis, suggesting commonality of guidance mechanisms in mammalian spermatozoa. This study further substantiates the function of hyperactivation as a means to direct spermatozoa in guidance mechanisms. The authors have no conflict of interest and no funding to declare.

Relationship between hyperactivation and intracellular calcium of human sperm.

Objective: Hyperactivation is recognized as a part of the process of capacitation. We previously reported that intracellular calcium of human sperm increased during capacitation, and calcium influx by progesterone was enhanced after capacitation. We have also presented data suggesting that the percentage of hyperactivated spermatozoa was greater in sperm populations exhibiting good versus poor fertilization at IVF-ET. In this study, we further analyzed the relationship between the percentage of hyperactivated sperm and intracellular calcium of human sperm.

Effects of cotinine on sperm motility, membrane function, and fertilizing capacity in vitro.

We evaluated the effect of cotinine on sperm fertilizing capacity in vitro. Human spermatozoa were washed and re-suspended in medium containing albumin and various concentrations of cotinine (0, 100, 200, 400, or 800 ng/ml). After an 8-h incubation period, sperm motility, hypoosmotic swelling test (HOST) outcome, and the percentage of hyperactivated spermatozoa were assayed. Aliquots of spermatozoa were then processed for the zona-free hamster oocyte sperm penetration assay (SPA) or hamster ooplasmic injections. Spermatozoa exposed to concentrations of cotinine equal to 400 or 800 ng/ml demonstrated significantly smaller outcomes for all of the above with the exception of after hamster ooplasmic injections, where high cotinine concentrations did not affect sperm viability or sperm capacity to undergo decondensation and activate hamster oocytes. It appears that cotinine concentrations of 400 or 800 ng/ml exert a detrimental effect on sperm motility, membrane function, and the ability to undergo capacitation. In addition, the current findings suggest that smokers with a high seminal plasma cotinine concentration who participate in assisted reproduction programs may be treated with intracytoplasmic sperm injections (ICSI) rather than conventional in vitro fertilization (IVF) trials.

Objective evaluation of hyperactivated motility in rat spermatozoa using computer-assisted sperm analysis.

The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks ( approximately 100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.

Diagnostic accuracy of computer-assisted sperm motion analysis.

One retrospective and two prospective studies were conducted among 218 couples treated with in-vitro fertilization (IVF) to establish the reproducibility and diagnostic accuracy of computer-assisted sperm analysis (CASA) with swim-up spermatozoa for the prediction of the fertilization rate of oocytes in vitro. Based on the results of a preliminary retrospective analysis in 49 patients, the 'curvilinear velocity' (VCL) was chosen as the most distinctive motion parameter of sperm function and the median was used to represent the entire sperm population. The number of inseminated motile spermatozoa was then adjusted to median VCL during two subsequent prospective studies with clinical IVF. Whereas in the first prospective study (90 couples) the threshold values of VCL with regard to the number of spermatozoa inseminated were based on the results of the preliminary retrospective study (49 couples), in the second prospective study (79 couples) the settings were based on the results of the first prospective study. The reproducibility of CASA was tested by analysing the motion characteristics of spermatozoa at different intervals after termination of swim-up, by repeated analysis of the same video-recording of the incubated spermatozoa by different observers, and by the repeated video-recording of the freshly prepared sperm samples and analysis of both video-recordings by the same observer. Under these conditions the frequency of disagreement between two measurements varied between 2.0 and 8.2%. In both prospective studies the sensitivity of CASA for the prediction of fertilization was high (74.0%), whereas the specificity was low (40.0%). In contrast to successful fertilization, unsuccessful fertilization of oocytes in vitro could not be predicted reliably with CASA. However, the pregnancy rate per cycle among patients with predicted low fertilization rates was significantly lower (5.3%) than in couples with high predicted fertilization rates (24.3%, P < 0.001). Therefore, CASA of washed spermatozoa may still help to identify couples who would benefit more from intracytoplasmic sperm injection (ICSI) than from IVF. A definite threshold level could not be identified for any of the motion parameters to distinguish the motion characteristics of fertilizing and non-fertilizing spermatozoa. Using various algorithms for hyperactivated motility, the percentage of hyperactivated spermatozoa was significantly higher among the successfully fertilizing patients than among the nonfertilizing group. However, the absolute number of hyperactivated spermatozoa added to the oocytes was higher in non-fertilizing couples. Therefore, the lack of fertilization in some patients may be caused by a generalized defect in sperm function rather than by insufficient hyperactivation.

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics.

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF-induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.

The potential shortcomings of measuring hyperactivated motility by computer-aided sperm analysis when sperm motion is multiphasic.

This paper was written from the standpoint that computer-aided sperm analysis (CASA) instruments, which capture a 'snapshot' of sperm trajectories in order to generate their data, may provide a poor measure of hyperactivated motility in a sperm population where hyperactivation is multiphasic in nature. To illustrate this point, a series of theoretical sperm populations were constructed which varied subtly but significantly in the nature of the hyperactivated behaviour expressed by spermatozoa. The parameters which were manipulated were: (i) the number of hyperactivated phases exhibited within a given period of time; (ii) the duration of these phases; and (iii) proportion of spermatozoa within the population which exhibited hyperactivated phases. These populations were then subject to an analysis in which snapshots of sperm motion were examined to determine the percentage of hyperactivated spermatozoa for that population. The results indicated that whilst this snapshot approach to quantifying hyperactivation could provide a figure for the percentage of hyperactivated spermatozoa within the sample window, this often inaccurately described the underlying behaviour of the population. Since there is very likely to be a significant amount of biological information contained within the nature of multiphasic behaviour, this paper has argued that this aspect of snapshot analysis is one which requires serious consideration by CASA manufacturers and medical researchers.

Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm.

Progesterone (P) has been reported to modulate numerous sperm functions through the binding of P to plasma membrane. One of the effects is an increase in sperm hyperactivation, which is known to be cAMP-dependent. To evaluate the effect of P on cAMP levels, human spermatozoa were incubated 2 h with increasing P concentrations. P significantly induced cAMP increase in a dose-dependent manner, reaching a 3-fold increase at 100 micromol/L (P < 0.01). During the study of the kinetics of P effect, two cAMP peaks were observed: one occurring after a 30-min incubation, with a 1.5-fold increase (P < 0.05), and the second one after a 120-min incubation, with a 2.5-fold increase (P < 0.01). These effects of P on cAMP levels correlated with significant rises in the percentage of hyperactivated spermatozoa, occurring at the same times as those of cAMP. To evaluate the Ca++-dependence of these P effects, the experiments were performed in the presence of and in the absence of Ca++ in the incubation medium. The effects of P at the 30th min and the 120th min were completely abolished in the absence of Ca++. Moreover, calcium ionophore A23187, after a 30-min incubation, induced an increase in cAMP levels identical to that obtained with P. The effect of P was partially reproduced by gamma-amino-butiric acid (GABA) and inhibited by GABA antagonist picrotoxin. It was also inhibited by tyrosine kinase inhibitor genistein but not by RU486. Based on these findings, we conclude that P induces Ca++-dependent cAMP increase in human sperm, that this effect is likely caused by the influx of Ca++ (previously reported), and that the effect partially involves GABA(A)-like receptors.