The effect of 12-myristate 13-acetate phorbol ester on human sperm hyperactivation

Although several dextran-coated iron oxide preparations are in preclinical and clinical use, little is known about the mechanism of uptake into cells. As these particles have been shown to accumulate in macrophages and tumor cells, we performed cellular uptake and inhibition studies with a prototypical monocrystalline iron oxide nanoparticle (MION). MION particles were labeled with fluorescein isothiocyanate or radioiodinated and purified by gel permeation chromatography. Two preparations of MION particles were used in cell experiments: nontreated MION and plasma-opsonized MION purified by gradient density purification. As determined by immunoblotting, opsonization resulted in C3, vitronectin, and fibronectin association with MION. Incubation of cells with fluorescent MION showed active uptake of particles in macrophages both before and after opsonization. In C6 tumor cells, however, intracellular MION was only detectable in dividing cells. Quantitatively, 125I-labeled MION was internalized into cells with uptake values ranging from 17 ng (in 9L gliosarcoma) to 970 ng iron per million cells for peritoneal macrophages. Opsonization increased MION uptake into macrophages sixfold, whereas it increased the uptake in C6 tumor cells only twofold. Results from uptake inhibition assay suggest that cellular uptake of nonopsonized (dextran-coated) MION particles is mediated by fluid-phase endocytosis, whereas receptor-mediated endocytosis is presumably responsible for the uptake of opsonized (protein-coated) particles.     

Sperm motility and morphology as changing parameters linked to sperm count variations

Variations in semen analyses of 177 males over a 1 year period were assessed. The average means of total counts, motility, morphology, total motile count and non-motile % were determined for 5 classes of patients ranging from azoospermic to normospermic. Positive relationships between a falling sperm count, a decrease in motility and total motile counts were seen. Also, increasingly, abnormal forms were found with lower sperm counts.     

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability

PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.     

Effects of pentoxifylline on sperm motility and hyperactivated motility in vitro: a preliminary report

Previous reports (2, 3) have suggested that pentoxifylline increases sperm motility. In this preliminary report based on five asthenozoospermic and five normal motility semen samples, we were unable to demonstrate any statistically significant effect of pentoxifylline on percent motility of human spermatozoa. However, in vitro exposure to capacitation medium with pentoxifylline may lead to an increase in total hyperactivated motility in asthenozoospermic samples, an effect not evident in the normal motility samples in this study.     

Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation

OBJECTIVE: To analyze the effect of sodium nitroprusside, a nitric oxide releaser, on sperm motion and lipid peroxidation-induced membrane damage in cryopreserved human sperm. DESIGN: Post-thaw, cryopreserved, human sperm samples were washed and divided into three aliquots. Each aliquot was incubated with either 0, 50, or 100 nM sodium nitroprusside. INTERVENTIONS: Samples were analyzed for lipid peroxidation (measured by malonaldehyde-thiobarbituric acid reactivity) at 3 hours post-thaw. MAIN OUTCOME MEASURES: Percent viability and motion parameters were assessed at 0, 10, and 30 minutes and 2, 3, 5, and 6 hours post-thaw. RESULTS: All results represent a mean +/- SEM, n = 10. Lipid peroxidation in samples incubated with 50 nM sodium nitroprusside (15.1 +/- 2.1 nM malonaldehyde/10(8) sperm) or 100 nM sodium nitroprusside (13.2 +/- 2.1 nM malonaldehyde/10(8) sperm) was significantly lower than in controls (22.7 +/- 3.1 nM malonaldehyde/10(8) sperm). Percent viability was significantly reduced from 0 minutes (60.6% +/- 3.5%) to 6 hours post-thaw in controls (38.0% +/- 5.1%) but not in 50 nM (46.8% +/- 10.4%) or 100 nM (48.8% +/- 6.5%) sodium nitroprusside-treated samples. Compared with controls (18.3% +/- 3.4%), maintenance of percent motility at 3 hours post-thaw was significantly improved in 50 nM (24.5% +/- 2.9%) and in 100 nM (26.3% +/- 3.2%) sodium nitroprusside-treated samples. Straight line velocity maintenance was significantly improved in 50 nM (37.3 +/- 1.3) and in 100 nM (37.0 +/- 1) sodium nitroprusside-treated samples as compared with controls (30.5 +/- 1.7). Significant improvements in curvilinear velocity maintenance compared with controls (56.3 +/- 2.9) also were observed in 50 nM (65.9 +/- 2.1) and 100 nM (72.1 +/- 4.1) sodium nitroprusside-treated samples. Significant differences in the motion parameters of sodium nitroprusside-treated samples were maintained at 5 and 6 hours post-thaw in comparison to controls. CONCLUSION: These results suggest that sodium nitroprusside is beneficial to the maintenance of post-thaw sperm motion and viability for up to 6 hours and that reduction of lipid peroxidative damage to sperm membranes may be the mechanism for these benefits.     

Effect of methylene blue, indigo carmine, and Renografin on human sperm motility

OBJECTIVES: To evaluate the effect of commonly used intraoperative vasography and tissue staining agents, indigo carmine, methylene blue, and Renografin, on sperm motility. METHODS: Semen from 20 healthy men was obtained after 2 to 4 days of abstinence. Sperm motility was initially evaluated in each specimen. Standard solutions of indigo carmine, methylene blue, and Renografin-60 were diluted 2x and 4x with lactated Ringer's solution. Equal aliquots of sperm were mixed with undiluted and diluted drugs, and sperm motility was assessed. RESULTS: Initial mean sperm motility was 70.3%+/-3.0%. Undiluted methylene blue and Renografin severely depressed sperm motility to 1.1%+/-0.5% and 2.3%+/-0.7%, respectively (P <0.05). Diluted methylene blue depressed motility to 4.9%+/-1.8% and 11.2%+/-3.0% (P < 0.05). Diluted Renografin depressed motility to 25.1%+/-4.1% and 55.3%+/-3.3% (P < 0.05). Although undiluted and 2x-diluted indigo carmine moderately decreased sperm motility (48.9%+/-3.2% and 61.7%+/-3.0%, P < 0.05), 4x-diluted indigo carmine had minimal effect on sperm motility (67.3%+/-2.8%, P > 0.05). Lactated Ringer's solution had no effect on sperm motility. CONCLUSIONS: We found a severe, immediate reduction in sperm motility after exposure to undiluted standard solutions of methylene blue and Renografin. Dilution of Renografin significantly decreased its negative impact on the sperm motility, whereas the adverse effect of methylene blue remained fairly constant even with increasing dilution. Sperm motility should be assessed prior to application of these agents. Sperm should be aspirated for immediate use and/or cryopreservation prior to the use of these agents. Indigo carmine may be safely used as a tissue stain or vasography agent with a minimal effect on sperm motility in dilutions of 4x and higher.     

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia

The Yugoslavia Prospective Study of environmental lead exposure has studied the associations between exposure to lead and pregnancy outcomes; childhood neuropsychological, behavioral, and physical development; and hematologic, renal, and cardiovascular function. The cohort comprises 577 children born to women recruited at midpregnancy in two towns in Kosovo, Yugoslavia; one town is the site of a lead smelter, refinery, and battery plant and the other is 25 miles away and relatively unexposed. A sample of these children has been followed at 6-month intervals through 7.5 years of age. Blood lead concentrations ranged from 1 to 70 microg/dl. Exposure to lead was not associated with adverse pregnancy outcomes. Exposure was associated with modest decrements in intelligence, small increases in blood pressure, higher risks of proteinuria, small increases in behavior problems, and perturbed hematopoiesis. Only at low level exposures (i.e., <16 microg/dl) were small associations with decreased height found. We discuss methodological problems that may hinder causal interpretation of these data, namely, use of blood lead concentration as an exposure measure, confounding, and town-specific associations. We conclude that while reported associations are small, collectively they lend support to the notion that lead is a toxicant with numerous adverse health effects.     

The in-vitro effects of nicotine and cotinine on sperm motility

Swim-up spermatozoa from the seminal samples of non-smokers, usually not exposed to passive smoking, were treated in vitro with nicotine (NIC) and cotinine (COT) at the average levels found in smokers' seminal plasma and at levels 500 times higher than this average. This was done to evaluate the action of these drugs on sperm motility. Each sample was allowed to swim up in Tyrode's solution with or without the drug; the study was carried out at time 0 and +1, +2, +4, +8 and +24 h of incubation, using a light microscope and a CASA system (experiment 1). In addition, the direct action of smoke on spermatozoa was studied using aspirated cigarette smoke (experiment 2). Kinetic parameters were then measured at 30 min, 45 min and 60 min starting from the last smoke injection. The first experiment showed that NIC and COT at average levels did not produce statistically significant variations of the kinetic parameters studied up to 24 h. However, the much higher concentration significantly altered all the kinetic variables in relation to the time of incubation. The second experiment with smoke in toto demonstrated a sharp reduction in all the sperm kinetic parameters. This reduction was seen after 30 min exposure to smoke and increased progressively until almost complete immotility at 1 h of exposure. These results suggest that NIC and COT are not responsible for the harmful effects of cigarette smoke on sperm kinetic parameters reported in the literature.     

The effect of timing and spatial separation on the velocity of auditory apparent motion

Previously, it was shown that the minimum conditions for the illusion of auditory apparent motion (AAM) depend on stimulus timing but not spatial separation. In the present experiment, the effects of stimulus timing and source separation on the perceived velocity of AAM were examined. Eight listeners estimated the velocity, duration, and distance traveled of AAM, using a no-modulus, magnitude estimation procedure. Four burst durations (25, 50, 100, and 300 msec), 10 stimulus onset asynchronies (SOAs; 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 msec) and two separations (10 degrees and 40 degrees) were tested. Perceived velocity estimates were related to the total duration (burst duration + SOA) of the stimulus sequence. The effect of separation on velocity was extremely small but statistically significant. These results are similar to those obtained previously on the minimum conditions for AAM. Duration estimates were related only to total duration, but separation estimates were related to both separation and total duration. These results suggest that velocity is possibly a primary dimension of AAM that is independent of source separation.     

Effects of alprostadil and prazosin on motility, viability and membrane integrity of human sperm

PURPOSE: We evaluated the effects of alprostadil, prazosin hydrochloride, and alprostadil/prazosin hydrochloride, agents used in the clinical treatment of male erectile dysfunction, on the motility, viability and membrane integrity of human sperm. MATERIALS AND METHODS: Ten healthy volunteers provided semen samples that were incubated with 0.4 mg./ml. alprostadil, 0.1 and 0.2 mg./ml. prazosin hydrochloride and 0.4 mg./ml. alprostadil plus 0.1 mg./ml. prazosin hydrochloride for 2 hours. Control incubations included polyethylene glycol 1450, the formulation vehicle for the clinical use of alprostadil and prazosin, and Ham's F-10 buffer. Serial evaluations of percent sperm motility, percent viability, membrane function (by hypo-osmotic swelling test) and several computer generated measurements of sperm motion, including straight line velocity, curvilinear velocity, linearity and amplitude of lateral head displacement, were made. RESULTS: None of the agents had a significant impact on the percentage of motile or viable sperm or on sperm membrane function. Incubation with 0.2 mg./ml. prazosin reduced straight line velocity and curvilinear velocity significantly compared with the other agents. These changes were most likely a direct result of the viscosity of the 0.2 mg./ml. prazosin solution and not a cellular or metabolic effect on the sperm. CONCLUSIONS: Alprostadil and prazosin hydrochloride at doses used in transurethral therapy for erectile dysfunction have no effect on the motility, viability and membrane integrity of human sperm.     

Comparison of different methods for the recovery of horse oocytes

The object of this study was to compare 4 different methods of oocyte recovery from mares; 1) transvaginal follicle aspiration in vivo; 2) follicle aspiration in vitro; 3) oocyte recovery by isolation of follicles in vitro and 4) follicle scraping in vitro. Oocyte recovery was highest after follicle scraping (71.1%) and follicle isolation and rupture (61.3%). Follicle aspiration in vitro and in vivo yielded oocytes on 31.2% and 19.3% of occasions, respectively. The output of different types of cumulus-oocyte-complexes was different among the methods; the portion of compact cumulus-oocyte-complexes was significantly higher with follicle scraping (50.7%) and follicle isolation (44.5%) than with aspiration in vivo (31.9%) and in vitro (23.7%). The recovery rate of oocytes from small follicles (<15 mm) was significantly higher than from larger follicles (P<0.05) using transvaginal follicle aspiration. The proportion of oocytes that were degenerate (exhibited shrunken, dense or visibly damaged ooplasm) ranged from 1.2% after follicle scraping, to 17.2% after aspiration in vivo. These results indicate that, for the recovery of horse oocytes in vitro, follicle scraping and follicle isolation give the highest recoveries of cumulus-intact oocytes.     

The effects of coital lubricants on sperm motility in vitro

Infertility affects approximately 15% of couples, and in about one-third the primary cause is a male factor. Patients undergoing infertility investigations frequently experience sexual dysfunction, which often is due to inadequate vaginal lubrication. This can lead to increased use of coital lubricants. The effects of such lubricants on sperm motility have not been widely studied, although sperm motility is one of the best prognostic indicators of fertilization. Using a prospective longitudinal control-based study, we analysed the effect of adding four lubricants: KY jelly, baby oil, olive oil and saliva on sperm motion in 16 samples from patients undergoing infertility investigations. Sperm samples were prepared by density gradient centrifugation prior to mixing with lubricants. Motility parameters were determined using computer-assisted semen analysis after 5, 15 and 30 min. All lubricants except baby oil significantly decreased percentage progressive motility, progressive velocity, curvilinear velocity and lateral head displacement at 12.5% concentration. At a lower concentration of 6.25%, both olive oil and saliva still significantly reduced progressive motility parameters, while KY jelly diminished head movement parameters. Hence, even at these very low concentrations, coital lubricants impair sperm motility and thus may adversely affect fertility.     

Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility

Tyrode fluid and Tyrode fluid plus Pentoxifylline were individually added to aliquots of semen samples obtained from 6 normal men and 6 infertile patients considered to have idiopathic normogonadotropic oligoasthenozoospermia. Pentoxifylline was added to final concentrations of 0.15, 0.30 and 0.60 mM. One aliquot with no addition served as control. Samples were incubated in 37 degrees C and observed by light microscopy at 30 minutes and at 1, 2 and 4 hours after obtaining the material. At observation time, semen quality was evaluated by determining the percentages of forwardly progressive spermatozoa, slowly progressive spermatozoa, "in situ" motile spermatozoa, live and non-motile spermatozoa and dead spermatozoa. Results reported included only the first and last category. Tyrode fluid did not affect significantly the motility and the duration of activity of spermatozoa. Ejaculated human spermatozoa both from normal and asthenozoospermic men added the Pentoxifylline at 0.30 and 0.60 mM showed a longer lasting activity than those of control semen and semen added only with Tyrode fluid.     

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization

BACKGROUND AND PURPOSE: We evaluated sucralfate, well-known in the treatment of gastric ulcers, in relation to its possible reduction of radiation-induced acute complications in the treatment of head and neck cancers. MATERIALS AND METHODS: One hundred two patients were randomized in a double-blind placebo-controlled prospective setting. All patients were treated to a minimum dose of 55 Gy in 5 weeks. Oral intake of sucralfate was started at the beginning of radiotherapy and continued during the whole treatment at a dose of 1 g six times a day. All patients were scored according to a scoring system developed in our department. Weight was checked once a week. RESULTS: Comparing the time course of the mean scores for subjective intolerance, mucositis, dysphagia, dermatitis and nausea, no statistically significant differences between the two treatment arms (sucralfate, n = 38; placebo, n = 45) were observed. The mean weight loss in the sucralfate arm was 1.6 +/- 3.4 kg while it was 1.3 +/- 2.0 kg in the placebo arm. Apart from gastrointestinal upset, the administration of sucralfate did not cause any side-effects. CONCLUSION: This trial produced no clinical evidence indicating that the oral intake of sucralfate reduces the acute radiation-induced side-effects. Therefore, we do not recommend the prophylactic use of sucralfate in patients with head and neck cancer treated by radiotherapy.     

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by magnesium and manganese

Because Mg2+ and Mn2+ are potent stimulators of motility through the stimulation of adenylate cyclase activity, the current study was undertaken to modulate the fertilizing ability of bovine semen by incorporation of various concentrations of those two salts in extenders before freezing. Motility analysis at 6 h in vitro showed a positive effect of MgCl2 in a dose-dependent manner from 0.5 to 5 mM (31 to 50%). Manganese at the concentration of 0.1 mM also supported good sperm motility (53%) compared with that of the control (28%). Although survival was increased, no detrimental effects were seen on the number of sperm that penetrated mucus of cows in estrus. The intracellular Ca2+ concentration of sperm was very different across treatments after thawing; spermatozoa that were extended with 2 mM MgCl2 and 0.5 mM MnCl2 possessed the highest concentrations at thawing. Four hours later, in the presence of Ca, spermatozoa that were extended in 0.1 mM MnCl2 showed the highest uptake. In the presence of Ca and heparin, spermatozoa that were extended in different amounts of Mg showed Ca2+ concentrations that increased in a dose-dependent manner. This effect was negated by glucose. Functional fertilizing capacity was also evaluated by in vitro fertilization, and the different treatments did not show any detrimental effects. In summary, 5 mM MgCl2 and 0.1 mM MnCl2 both have beneficial effects for the maintenance of sperm motility without detrimental effects on mucus penetration and fertilizing ability. Furthermore, these treatments do not prevent subsequent Ca2+ uptake in response to heparin. These in vitro studies are potentially a good sorting system to predict the benefits of extender modifications.     

Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization

OBJECTIVE: To look for correlations between acridine orange (AO) staining and semen parameters before and after sperm separation procedures and to assess whether the AO test predicts fertilization or pregnancy outcomes after standard IVF and intracytoplasmic sperm injection. DESIGN: Prospective study that simultaneously assesses sperm morphology and nuclear protein maturity on a cell-by-cell basis before and after preparative procedures. SETTING: University teaching hospital. PATIENT(S): Men (n = 140) undergoing diagnostic semen analysis. MAIN OUTCOME MEASURE(S): Acridine orange fluorescence of sperm nuclei, semen parameters, IVF outcome. RESULT(S): In unprocessed samples, 90% of sperm with normal heads displayed green fluorescence (mature nuclear protein); significantly lower percentages of green fluorescence were observed in sperm with abnormal heads. The percentage of mature normal sperm in the specimen correlated with motility. Sperm maturity after swim-up or Percoll gradient was significantly improved for sperm with normal or abnormal heads. The percentage of mature normal sperm correlated with motility after either Percoll or swim-up. Neither the percentages of mature nuclei nor mature normal nuclei correlated with fertilization or pregnancy outcome. CONCLUSION(S): Nuclear protein maturation correlates with sperm motility and morphology. Because morphologically normal and motile sperm are more mature, separation procedures should generate a population of sperm with the highest fertilization capacity. Acridine orange staining, however, did not predict fertilization efficiency or pregnancy outcome in IVF cycles.     

Complementary effects of propranolol and nonoxynol-9 upon human sperm motility

The inhibitory effects of nonoxynol-9, DL- and D-propranolol upon human sperm motility were determined in vitro. All three compounds were capable of causing complete cessation of sperm movement. However, greater efficacy was achieved using combinations of nonoxynol-9 and propranolol, suggesting a complementary interaction between these compounds. Investigations of the mechanism of action of propranolol revealed that an influx of calcium accompanied the loss of motility. However, since incubation in the absence of calcium enhanced the spermicidal effects of this compound, it was concluded that this calcium influx did not constitute the primary means by which motility was disrupted. Low doses of propranolol, which did not affect motility, were found to inhibit the capacity of human spermatozoa for sperm-oocyte fusion.     

Comparison of two methods of surfactant administration and the effect on dosing-associated hypoxemia

OBJECTIVE: The purpose of our study was to test the hypothesis that surfactant dosing through a proximal sideport adapter on an endotracheal tube leads to more dosing-associated hypoxemia compared with a method of dosing that uses a double-lumen endotracheal tube. STUDY DESIGN: Using adequate sample size to compare significant changes in O2 saturation (power > 0.8, alpha < 0.05) we enrolled 36 infants with respiratory distress syndrome in this randomized trial. A 10% change in O2 saturation was considered clinically significant. Nineteen infants received 38 doses of surfactant through the sideport adapter. Seventeen infants received 31 doses of surfactant through the nonventilation lumen of a double-lumen endotracheal tube. Two main outcome measures were assessed: time-averaged O2 saturation values 30 minutes after dosing and the largest absolute fall in O2 saturation for each patient. RESULTS AND CONCLUSIONS: Time-averaged O2 saturation measures were higher in the proximal sideport group (p = 0.02), but the magnitude of difference was probably not clinically significant. No significant difference was detected between groups when we compared largest absolute drop in O2 saturation. Secondary analyses found no effect of birth weight or dose number (second vs third dose) on either outcome measure.     

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by female genital products

Because the different portions of the female genital tract act in many ways on sperm metabolism, the current study was undertaken to modulate the survival and fertilizing ability of bovine semen by incorporation of products from the oviduct or the follicle in extenders before freezing. Motility rates at 6 h in vitro showed a net positive effect when biological factors from total retentate or from a fraction of bovine follicular fluid (total retentate = 43%; fraction 2 = 54%), oviductal cell culture (total retentate = 43%; fraction 2 = 58%), or granulosa cell culture (total retentate = 43%; fraction 3 = 53%) were added to the extenders compared with the addition of BSA (31%). Fraction 3 of granulosa cell culture retentate also had a significant stimulatory effect on the number of sperm that penetrated mucus of cows in estrous compared with BSA (n = 205 vs. n = 159). The intracellular sperm Ca2+ concentrations were very different across treatments after thawing. Sperm from straws with BSA had the highest concentration. At 4 h, intracellular Ca2+ concentration increased for all treatments, except that for sperm treated with BSA and Ca alone, internal Ca2+ declined. Heparin plus Ca stimulated a greater internalization of Ca2+ than did Ca alone for retentate from bovine follicular fluid, oviductal cell culture, and BSA treatments: glucose consistently and significantly reduced internalization. In vitro fertilization rates were similar, and no significant differences were observed across treatments.