Human Sperm Characteristics Research Articles

P-090 The in vitro motility characteristics of human sperm collected by microfluidic device

Abstract Study question Do the in vitro motility characteristics of sperm collected using a microfluidic device (ZyMot) differ from those of sperm collected by density gradient centrifugation (DGC)? Summary answer ZyMot-collected sperm had longer-lasting motility than those collected by DGC, but the head beat-cross frequency was inferior due to the absence of washing in vitro. What is known already ZyMot (DxNow, Inc., USA) can sort highly motile sperm with low reactive oxygen species (ROS), and DNA damage, without centrifugation. In IUI cycles, although the total number of ZyMot collected sperm was lower than that of DGC, it was reported that the pregnancy rate of ZyMot cycles was higher than that of DGC cycles. Washing sperm with a medium is essential for in vitro capacitation; however, the protocol for microfluidic devices lacks this procedure. The aim of this study was to clarify the motility characteristics of ZyMot-collected sperm. We also examined how washing ZyMot-collected sperm affected motility. Study design, size, duration The specimens were provided by patients who underwent semen analysis between October 2022 and January 2023, with written consent. We used 12 samples with a semen volume of 2.5 ml or more, or a motile sperm concentration of 4 million/ml or more. The same donor samples were divided into three groups (DGC, ZyMot, and ZyMot with wash). Dunnett’s test was performed with the DGC group as the control group. Participants/materials, setting, methods In the DGC group, 2-layer DGC (400G, 20 min) was performed, followed by washing (400G, 5 min). In the ZyMot group, ZyMot was used according to the manufacturer’s protocol. In the ZyMot with wash group, ZyMot-collected sperm was centrifugally washed (300G, 5 min). All samples were cultured at 30 °C until motility analysis. Sperm motility was analyzed using a computer-assisted sperm analysis system (SMAS) after 0, 4, and 24 h of semen preparation. Main results and the role of chance There was no significant difference in the motility rate of the two ZyMot groups compared to that of the DGC group at 0 and 4h after sperm preparation. On the other hand, the motility of the ZyMot group tended to be higher (53.3%, p = 0.0787), and the motility of the ZyMot with wash group was significantly higher (85.4%, p < 0.001) than that of the DGC group (33.5%), after 24h of sperm preparation. The straight-line velocity, curvilinear velocity, and average speed of the ZyMot and ZyMot with wash group did not differ from those of the DGC group at 0 and 4h. All speed parameters of the ZyMot with wash group were significantly higher than those of the DGC group, after 24h of culturing. Regarding sperm head movement, there was no significant difference in the amplitude of lateral head displacement between the two ZyMot groups compared to that of the DGC group at 0 and 4h of culture. In contrast, the sperm head beat-cross frequency of the ZyMot group was lower than that of the DGC group (9.18±0.31 Hz vs 8.71±0.49 Hz, p = 0.0409). Both head movement parameters of the ZyMot with wash group were significantly higher than those of the DGC group. Limitations, reasons for caution We chose semen samples with a high number of motile sperm because we divided the sperm into three experimental groups. Therefore, it is unclear whether our findings are applicable to patients with poor semen parameters. Wider implications of the findings ZyMot may be advantageous for IUI cycles because ZyMot-collected sperm maintains sustainable motility for extended periods. Alternately, ZyMot may be unfavorable compared to DGC in conventional IVF cycles due to inferior sperm head movement, which is a marker of capacitation. However, this disadvantage can be overcome by washing. Trial registration number not applicable

Effect of pesticide exposure on human sperm characteristics, genome integrity, and methylation profile analysis.

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group.

P-022 Relationship between sperm movement characteristics assessed by a sperm motility analysis system (SMAS) and normal fertilization rates in vitro

Abstract Study question Is there a relationship between movement characteristics of human sperm assessed by SMAS and normal fertilization rates in vitro? Summary answer The curvilinear path wobble of motile sperm before insemination is predictive of normal fertilization rates after in vitro fertilization (IVF). What is known already Assessment of semen parameters is very important in both the diagnosis of male infertility and the selection of methods for treatment with conventional-IVF (C-IVF) or intracytoplasmic sperm injection (ICSI). Several studies have reported that objectively movement characteristics of motile sperm assessed by SMAS are related to the functional capacity of human sperm. However, there are no established the objective indicators and criteria for sperm motility characteristics assessed by SMAS. In this study, we investigated the utility of the movement characteristics of motile sperm measured by SMAS as an indicator of normal fertilization rates. Study design, size, duration Retrospective experimental trial conducted in a single IVF center between January 2018 and February 2020. Sperm movement characteristic analysis was performed using the SMAS in 300 IVF cycles using sperm from 270 men. At least three metaphase II oocytes were inseminated in C-IVF. The SMAS estimates were measured in semen and before insemination. This study was approved by the Ethics Committee of the Institutional Review Board (IRB) of Kinutani Women’s Clinic, Hiroshima, Japan. Participants/materials, setting, methods Motile sperm from at least 3 different fields were observed in each sample and their parameters were examined. First, we investigated the correlation between the normal fertilization rates and sperm motility characteristics. Next, the group comprising 300 cycles was classified into a group of a ≥ 60% normal fertilization (group 1: 229 cycles) and a group with <60% normal fertilization (group 2: 71 cycles) to assess the sperm characteristics described below. Main results and the role of chance The following indicators were evaluated: amplitude lateral head displacement (ALH), best cross frequency (BCF), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN=VSL/VCL), straightness (STR=VSL/VAP), and curvilinear path wobble (WOB=VAP/VCL).There were significant correlations between normal fertilization rates and semen parameters, including VCL (r = 0.014; P < 0.05) and ALH (r = 0.177; P < 0.05). However, there were no significant correlations for other parameters. Moreover, there were no significant correlations between normal fertilization rates and sperm parameters before insemination. The SMAS estimates in semen and sperm before insemination were compared between group 1 and group 2. There were no significant differences in most baseline characteristics across groups, except for the number of normally fertilized oocytes. However, there were significant differences in sperm WOB before insemination between group 1 and group 2 (0.589 vs. 0.572; P < 0.05). Sperm motility before insemination was more curvilinear in group 1 than in group 2. The optimal cut-off value for WOB of motile sperm before insemination for predicting≥60% normal fertilization was 0.587 (AUC was 0.60; P < 0.05). These results were suggested that the WOB of sperm before insemination is useful as an indicator in determining the selection of methods for fertilization in vitro. Limitations, reasons for caution The main limitations of this study are its retrospective design and small sample size. A prospective study is necessary to further clarify the prediction criteria. Wider implications of the findings This study suggests that the WOB of motile sperm before insemination may be used as one of the indicators to predict good fertilization rates after C-IVF. Trial registration number N.A.

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients

BackgroundCryopreservation of human spermatozoa has been identified as an efficient procedure to preserve fertility in men before any cancer therapy or surgical infertility treatment. Despite the benefits of the procedure, the deleterious effects of cryopreservation have been proven on sperm structure and function. This study aimed to evaluate seminal plasma effects on human sperm characteristics after cryopreservation, and compare the addition of normozoospermic and oligozoospermic seminal plasma in the prepared oligozoospermic samples. Semen samples were collected from fifty-five oligozoospermic men and the twenty fertile individuals who referred to the infertility center. At first, a semen analysis was carried out on each neat ejaculate, and then some were cryopreserved. The remainder of the semen was divided into two, one for seminal plasma removal and the other for sperm preparation. Then, the prepared spermatozoa were cryopreserved in three groups: one with, and another without the addition of oligozoospermic seminal plasma, and still another with the addition of normal seminal plasma. After thawing, sperm DNA integrity, viability, motility, and morphology were determined.ResultsThe percentages of all parameters were significantly lower after cryopreservation in all groups compared to the fresh sample. However, this reduction was lower in the oligozoospermic samples cryopreserved with normal seminal plasma.ConclusionThe results indicated that seminal plasma in oligozoospermic patients could not support sperm against cryo-injuries, an indication likely due to insufficient antioxidants and other protective components in oligozoospermic patients. However, normal seminal plasma could slightly preserve sperm characteristics after cryopreservation in oligozoospermic patients.

Sperm cryostorage in a dry tank: An accurate alternative

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at −194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

Ультраструктурні характеристики сперміїв людини після кріоконсервування шляхом вітрифікації з полівінілпіралідоном

Ультраструктурні характеристики сперміїв людини після кріоконсервування шляхом вітрифікації з полівінілпіралідоном

Human Sperm Characteristics with Regard to Cobalt, Chromium, and Lead in Semen and Activity of Catalase in Seminal Plasma.

We analyzed cobalt (Co), chromium (Cr), and lead (Pb) concentrations in human semen and catalase CAT activity in seminal plasma and the effects of their relations on the sperm quality. We obtained semen samples from men (n = 168) undergoing routine infertility evaluation. Studies included two groups based on the ejaculate parameters: I (n = 39; normal ejaculate; normozoospermia); II (n = 129; pathological spermiogram). We examined relationships and differences between Co, Cr, and Pb concentrations in seminal plasma, CAT activity, and semen parameters. We did not establish differences in Co, Cr, and Pb concentrations and CAT activity from men between normozoospermic and those with pathological spermiogram. We found a significantly lower Co concentration and CAT activity in males with normal sperm motility than in asthenozoospermic males. We found significantly lower Co and a higher Pb concentration in males with normal morphology of spermatozoa than in teratozoospermic males. We found a significantly higher Pb concentration in the individuals with consumption of alcohol than in those without consumption. There were significant correlations between Co and Pb concentrations, sperm progressive motility (A + B, i.e., fast and slow progressive motility; Co-negatively; Pb-positively), and normal morphology of spermatozoa (Co-negatively; Pb-positively). We found a significant negative correlation between Cr concentration and slow progressive motility, and between CAT activity and volume of ejaculate. Co, Cr, and Pb levels and CAT activity were related to sperm characteristics and male fertility. The impact of alcohol may be manifested by a disturbance in Pb equilibrium in the body. Co and Pb influence progressive motility and normal morphology of human spermatozoa. Thus, Co and Pb levels in semen may be a useful diagnostic in male infertility. Most of the results of this study are in contrast to expectations. Namely, Pb is a toxic element and its harmful effects (poor semen quality) may be expected already at relatively low level of Pb exposure and are particularly visible with increasing of Pb. Co and Cr(III) are essential elements and harmful effects may be expected at their deficiency and/or overexposure.

Silver nanoparticle induced toxicity to human sperm by increasing ROS(reactive oxygen species) production and DNA damage

Silver nanoparticles (AgNPs) have been used in medical products and industrial coatings, due to their antimicrobial properties. Excessive use of AgNPs can have adverse effects on the human body, however, their toxicity characteristics to human sperm and the potential mechanisms are not entirely clear. In this study, we exposed human sperm to different doses of AgNPs (0, 50μgml−1, 100μgml−1, 200μgml−1 or 400μgml−1) for various times (15min, 30min, or 60min), followed by analyses of the sperm viability, motility and the ratio of abnormal to normal sperm.Then, transmission electron microscopy(TEM) was used to explore the sperm ultrastructural characteristics. Reactive oxygen species production and DNA fragmentation were tested using standard kits and the sperm chromatin dispersion method, respectively. The results showed a dose- and time-dependent decline in sperm viability and motility and an increased ratio of abnormal to normal sperm after 30min and 60min of exposure to AgNPs at 200μgml−1 and 400μgml−1. The most common abnormalities were sperm heads with disrupted chromatin or absent acrosomes, bent tails, and curved mid-pieces. The ultrastructural characteristics of AgNP-treated sperm included disrupted, swollen, granular and vacuolar defects of the chromatin. In addition, ROS(reactive oxygen species)production and DNA fragmentation were markedly increased after 60min of exposure to AgNPs at 200μgml−1 and 400μgml−1. Our results indicated that AgNPs caused detrimental changes in human sperm characteristics, and the excessive use of AgNPs should be carried out with caution.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

P-183: A comparative study on TEST-Yolk buffer and human sperm preservation medium on post thaw characteristics of human sperm from pre-freeze specimens

Optimum cryosurvival is dependent not only on freezing methodologies, but also on the type of cryopreservatives used. It seems reasonable to speculate that removing the seminal plasma and selecting highly motile spermatozoa by an optimal sperm processing technique, before freezing, may reduce the loss of viability, motility and acrosome integrity during cryopreservation. Moreover, this method can remove those cells that produce free radicals thereby minimizing peroxidative damage during freeze-thaw procedure.

Correlation of the Bovine Cervical Mucus Penetration Test With Human Sperm Characteristics in 1406 Ejaculates

Correlation of the Bovine Cervical Mucus Penetration Test With Human Sperm Characteristics in 1406 Ejaculates

P-253 Correlation of the bovine cervical mucus penetration test with human sperm characteristics in 1406 ejaculates

P-253 Correlation of the bovine cervical mucus penetration test with human sperm characteristics in 1406 ejaculates

Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342

To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA). Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm. Normal human volunteers in an academic research and medical environment. Selected healthy student volunteers. Delivered semen to the laboratory within 1 hour of collection. Semen quality measured by CASA. Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent. The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.

Human sperm characteristics before and after preparation for in vitro fertilization

Human sperm characteristics before and after preparation for in vitro fertilization

Effects of propranolol and caffeine on movement characteristics of human sperm

Human sperm, separated on Percoll gradients and transferred to cell culture medium, were exposed to various concentrations of propranolol (0.8-800 microM) or caffeine (3 microM-17 mM) for 4 h. Their motility pattern was analysed after 5 min and 4 h, employing the computerized Cellsoft system. Curvilinear velocity (VCL), percentage motile sperm, linearity (LIN), mean amplitude of lateral head displacement (ALHMEAN) and beat cross-frequency (BCF) were assessed. Both drugs had a practically immediate effect on the sperm. Propranolol concentrations greater than 80 microM had a negative effect on all movement variables, except VCL and ALHMEAN, which showed a slight, non-significant, initial increase. LIN and the percentage motile sperm appeared to be somewhat negatively affected at lower concentrations (80 microM) than the other variables, and were reduced further at higher concentrations and with time. The presence of 800 microM propranolol immobilized all sperm within 4 h. Caffeine at 1.7 and 5 mM, increased VCL and ALHMEAN. In contrast, the highest caffeine concentration tested (17 mM) had a negative effect on all variables at 4 h after addition.

Effect of duration of abstinence on maturity of human spermatozoa nucleus.

Human sperm are a heterogeneous population, particularly with respect to their morphology, motility, and degree of nuclear maturity. The characteristics of human sperm and the degree of nuclear condensation with variable sexual abstinence times (long, 7 days; short, 12 h) have been studied. Long abstinence led to an increase in the number of sperm and a decrease in their motility, but their morphology remained unchanged. The DNA-protein complex demonstrated by ethidium bromide uptake was unchanged, but there was a significant increase in nuclear stability upon treatment with SDS. The duration of abstinence hardly affected the degree of nuclear condensation or stability of human sperm. The heterogeneity observed is essentially of testicular origin.

Human sperm characteristics during frequent ejaculation.

Sperm concentration, morphology, DNA condensation and nuclear protein pattern as well as sperm adenylate cyclase were evaluated in semen samples provided by 7 volunteers every 8 h for 2 days. During the observation period, sperm concentration and total sperm number decreased but began to return towards normal after a 3-day abstinence period. No increase in the proportion of immature sperm cells was observed by light microscopy. Total adenylate cyclase (EC 4.6.1.1) increased significantly per cell, due to an increase in the soluble component while the particulate component remained relatively constant. Microflow fluorometry revealed no consistent alterations in the DNA or nuclear protein distribution. We conclude that although a high frequency of ejaculation does not disturb the conventional measures of sperm integrity, such as DNA condensation, there are major changes in at least one biochemical measurement, the activity of soluble adenylate cyclase.

Motility characteristics of human sperm, nonfrozen and cryopreserved.

Ejaculates (1651) obtained by masturbation from 88 donors were evaluated for count, motility, and kinetics. After the initial evaluation the ejaculates were frozen in liquid nitrogen, thawed 24 hr later, and assessed for postthaw motility and kinetics. The ejaculates were then divided into seventeen groups according to sperm count and evaluated to determine if direct relationships exist between prefreeze and thawed semen characteristics. The average sperm count was 151 X 10(6) sperm/ml. The freezing process resulted in a reduction of motility from a mean of 85% to a mean of 52%. Postthaw motility increased with sperm count up to 120 X 10(6) sperm/ml where there appeared to be no further effect. Linear regression analysis demonstrated a high correlation between prefreeze motility and postthaw motility (r = 0.947). Cryopreservtion of human spermatozoa results in a significant reduction of motility. A direct relationship exists between prefreeze and postthaw motility and prefreeze motility may be an important factor in evaluating the potential of an ejactulate to withstand cryopreservation.