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.     

Use of nonoxynol-9 and changes in vaginal lactobacilli

The conformation of lens crystallins in vivo or in a highly concentrated solution is not well established. Most studies were carried out in dilute solutions in which protein-protein interaction is minimal. In order to see whether there is conformational change (tertiary and secondary structures) when crystallin solutions are brought to high concentrations, we have performed the following molecular spectroscopic measurements: circular dichroism (CD) and Fourier transform infrared (FTIR). Near-UV CD measurements showed a more than two-fold increase in CD intensity (molar ellipticity) for the total water-soluble (WS) protein from young calf lens nucleus in a highly concentrated solution (> 300 mg/ml in a 0.01-mm cell), when compared with a dilute solution (1000-fold dilution in a 10-mm cell). The individual crystallins in concentrated solutions also showed an increase in CD intensity, but of different magnitude: alpha-crystallin > beta-crystallin > gamma-crystallin. The increased CD indicates that lens crystallins are in a more compact structure in highly concentrated solutions; they likely undergo a transition from a mobile to an immobile state. Change in near-UV CD usually is caused by restricted mobility of aromatic side groups, particularly Trp. The transition involves not only a change in protein tertiary and/or quaternary structure, but also in protein backbone structure. The change of protein backbone structure was drawn from FTIR measurements. FTIR spectra, sensitive to the secondary structure in the amide I region, could be measured for a highly concentrated solution for which far-UV CD measurement is not feasible. The secondary structure that showed prominent change for alpha-crystallin in a highly concentrated solution was beta-conformation: increase in beta-turn with a concomitant decrease of alpha-helix structure.     

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.     

Computer-assisted analysis of sperm motility

The fully automatic method for reconstruction of spermatozoa trajectories was presented. The method is based on computer image analysis. It allow to process pure semen without any treatment like diluting or purifying. Since no human interacting is available, this method lead to objective results. The method was used to analyse semen taken from healthy men (WHO 92 standard). The result shows absence of different populations in single sample.     

Kobe earthquake and reduced sperm motility

We investigated a possible relationship between the Kobe earthquake (January 17, 1995) and the quality of semen. We assessed sperm concentration and motility of 27 male patients who had a concentration of more than 30 million/ml and >40% sperm motility within 5 months before the earthquake. Twelve male patients from districts with a magnitude of <4 on the Richter scale showed no difference in sperm concentration and motility before and after the earthquake. Of 15 male patients from districts with a magnitude of >6, five patients whose houses received no damage showed no distinct changes in sperm concentration and motility. In contrast, 10 patients whose houses were partially or completely destroyed showed significantly (P < 0.001) lower sperm motility after the earthquake than before, although no significant difference of sperm concentration could be observed. Of these latter 10 patients, seven could be followed. In six patients, sperm motility was restored between 2 and 9 months after the earthquake; the sperm motility in one patient, whose father died a victim of the house crash, has not yet recovered. Thus, the acute stress resulting from such a catastrophic earthquake could be a possible cause of reduced sperm motility.     

Self-reported discomfort associated with use of different nonoxynol-9 spermicides

Patients with the syndrome of chronic daily headache often report migrainous symptoms and consequently are diagnosed as having a primary headache syndrome. We report two cases of idiopathic intracranial hypertension causing chronic daily headache with migrainous features in the absence of associated papilledema.     

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.     

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.     

The effect of propranolol upon chick embryo cardiogenesis

The teratogenic effects of propranolol HCl on cardiac development were studied in chick embryos of days 3 and 4 of incubation. Propranolol was injected into the yolk sac at doses ranging from 0.05 to 0.6 mg per egg. All the treated and control embryos were examined on day 7. The LD50 for the embryos treated on the 3rd and 4th day was 0.15 and 0.35 mg per embryo, respectively. Cardiac anomalies such as aortic stenosis ventricular septal defects and common truncus arteriosus were observed. Other malformations included atrial septal defects, thin atrial wall and defects of the pulmonic, aortic and atrioventricular valves. The incidence of cardiac anomalies in the controls was very low. Propranolol was observed to slow the heart rate in the experimental embryos. It is suggested that slowing of heart rate at the early stages of heart development caused aberrant bloodstream flow patterns which probably resulted in the genesis of cardiac anomalies. The results of this study indicate that propranolol has teratogenic effects on chick embryo cardiogenesis.     

Cytogenetic effects of cryopreservation on human sperm: assessment using an improved method for analyzing human sperm chromosomes

OBJECTIVE: To evaluate any cytogenetic effects of cryopreservation on human sperm by comparing the frequencies of sperm chromosome anomalies and sex ratios before and after freezing. METHODS: Using in vitro fertilization of zona-free hamster oocytes, analysis of sperm chromosomes was first performed on portions of fresh human semen samples. The residual semen was then analyzed for sperm chromosomes after cryopreservation for several weeks. Sperm donors were 5 healthy men aged 26-38 years. RESULTS: A total of 166 sperm karyotypes were analyzed, 94 before freezing and 72 after freezing. The results indicated no significant differences between fresh and frozen sperm in either frequencies of aneuploidy (fresh: 0%, frozen: 2.8%) or structural anomalies (fresh: 7.5%, frozen: 9.7%). The sex ratios did not differ from the expected 1:1 ratio under either condition. CONCLUSIONS: The results of these studies indicate that cryopreservation does not exert any cytogenetic mutagenicity on human spermatozoa or alter X/Y ratio of human sperm.     

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.     

Effects of nonoxynol-9 on vaginal microflora and chlamydial infection in a monkey model

A 58-year-old male with left renal cell carcinoma and prostatic carcinoma occurring synchronously, is reported. He visited our hospital, because of the high level of serum prostate-specific antigen (PSA) pointed out in a health screening by his company. Prostatic cancer was detected in both lobes of the prostate by needle biopsy specimens and histopathology represented moderately > poorly differentiated adenocarcinoma. Magnetic resonance imaging (MRI) and computed tomography (CT) revealed no cancer invasion beyond the prostate and no lymph node metastasis. Bone scintigram showed no abnormal RI accumulation to bone. Therefore, his prostatic cancer was considered to be at stage B2. Abdominal ultrasound echogram showed the mass lesion in the left kidney. CT and angiogram also demonstrated a left renal tumor. Left radical nephrectomy was performed and histopathology showed a mixed subtype of renal cell carcinoma (stage: pT2b, pN0, pM0). Although 94 cases of double cancers associated with genitourinary organs have been reported in the Japanese literature, only 4 cases of double cancers of renal cell carcinoma and prostatic cancer have been reported.     

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.     

Development of sperm motility patterns in the murine epididymis

The maturation of sperm motility in the epididymis of the mouse was assessed using a computer-assisted sperm analysis system. Spermatozoa were immotile in the most proximal regions of the epididymis but developed motility rapidly in the proximal caput epididymis; the percentage motility remained high thereafter. In the caput, flagellar beat was erratic with little progression, but in the proximal corpus region circular movement patterns were reflected in reduced linearity (LIN) and straightness (STR) of the sperm tracks, although velocities were little changed and wobble (WOB) increased. In the mid-corpus region, however, all velocities, LIN, STR and WOB, increased markedly. In more distal regions there was little change in these parameters. Distribution curves of the kinematic parameters of spermatozoa obtained from each region indicated that the most heterogeneous population was that from the mid-corpus epididymis; the most homogeneous was that from the mid-cauda region. Individual sperm tracks revealed slowly progressing spermatozoa in the distal caput, transforming to motion in small circles, interrupted by more linear progression. More distally, linear progression was interrupted by looping movements and a generally progressive path was observed thereafter, with less deviation from the average path as the spermatozoa matured. Spermatozoa displaying motion compatible with passing the uterotubal junction were first found in the proximal corpus epididymis, in agreement with earlier in-vivo fertilization studies on where fertilizing capacity is achieved with epididymal spermatozoa.     

Bull sperm motility and membrane integrity in media varying in osmolality

Studies were designed to evaluate bull sperm motility and membrane integrity following exposure to Tyrode's solution varying in osmolality from 100 to 1537 mOsm. Congo red and bisbenzimide (HOECHST 33258) stains were used to distinguish between sperm with intact versus disrupted plasma membranes. Sperm motility was subjectively evaluated. No significant differences were found between motile and unstained sperm (sperm with intact membranes) in solutions with nearly physiologic osmolalities between 200 and 300 mOsm. However, in 100- and 150-mOsm solutions, the percentage of motile sperm (5 and 19%), respectively) was lower than the percentage of sperm unstained with Congo red (18 and 35%). With HOECHST 33258 stain, the corresponding values were 7 and 14% versus 26 and 31%. The percentage of motile sperm declined greatly in the 500-mOsm medium, but the proportion of unstained sperm was affected little until the osmotic pressure exceeded 732 mOsm. Partial recovery of motility occurred when sperm were returned to the isotonic medium. This study indicated that the sperm plasma membrane was more resistant to osmotic damage than were the mechanisms responsible for sperm motility. Lack of motility in hypertonic media was not an absolute indicator of cell death, and unstained sperm overestimated sperm viability, which are factors to consider when sperm are being evaluated, especially relative to cryopreservation.     

Purification and characterization of reversible sperm motility inhibitors from porcine seminal plasma

Although cigarette yields of tar and nicotine have been declining since the early 1970s, little information is available for the general population on the consequences of their use on exposure to tobacco combustion products. In a population-based sample of 298 smokers, the majority of whom were Hispanic, we examined the relationships between yields of cigarettes currently smoked and levels of salivary cotinine and end-expired carbon monoxide. Spearman correlation coefficients between the current number of cigarettes smoked and cotinine (r = 0.52) or carbon monoxide (r = 0.51) were higher than correlations between the Federal Trade Commission (FTC) nicotine data and these same markers, 0.12 and 0.05, respectively. Correlations between FTC tar and carbon monoxide yields and the biologic markers were similarly weak. In multiple linear regression models, the current number of cigarettes smoked was the most important predictor of cotinine and carbon monoxide levels (p < 0.0001). The addition of FTC tar, nicotine, or carbon monoxide to the models explained little of the variability in cotinine or carbon monoxide levels. Because FTC yields of tar and nicotine are poor predictors of exposure to tobacco combustion products, subjects' reports of cigarette brand should not be used as a primary marker of exposure in epidemiologic investigations. Furthermore, smokers need to be advised about the limitations of cigarette yield information for predicting the potential for adverse health effects of smoking.     

Genetics of human sperm

The mechanism by which low doses of methotrexate act in psoriasis to restore a clinically normal skin is poorly understood. Apoptosis is a programmed cell death activated when cell removal is needed. The purpose of the present work was to examine using an organotypical model of keratinocyte culture, the possibility that low doses of methotrexate can induce apoptosis of keratinocytes. Epidermal explants were cultivated on dead deepidermized dermis under air-exposed conditions. After 10 days, methotrexate (10(-7) M) was added. After a further 5 days, one part of each culture was fixed and submitted to routine histology, DNA nick end labelling (TUNEL) to detect DNA fragmentation (a molecular marker of apoptotic cell death) and immunohistochemical detection of p53 (a protein involved in apoptosis induced by DNA-damaging agents). The other part of each culture was processed for electron microscopy. A significant proportion of keratinocytes (1%) were damaged and exhibited the morphological features of apoptotic cell death. Immunohistochemical overexpression of p53 was detected in the basal layer of the cultures treated with methotrexate. Low doses of methotrexate induce apoptosis. This mode of action could explain the reduction in epidermal hyperplasia during treatment of psoriasis with methotrexate.