Male adolescent exposure to endocrine-disrupting pesticides: vinclozolin exposure in peripubertal rabbits.

Adolescence is a time of dramatic neuroendocrine changes that are required for sexual maturation. Hormonal mimicking or inhibiting chemicals can cause significant impairment during this critical period. Vinclozolin (Vin) has been shown to be an anti-androgen affecting male offspring in rats in utero, and its mechanism of action may be mediated by inhibition of androgenic receptor action. The majority of teenagers working on farms are male, and therefore a systemic fungicide, vinclozolin, was selected for study. The rabbit has proved to be an excellent species for modelling reproductive toxicant effects in the male and was selected as the test species. The peripubertal phase for the rabbit was determined to be between the 3rd and 4th months. A 2-month dosing period was therefore initiated at 3 months of age and carried through to the 4th month. Vin was administered by dermal application (100 mg kg(-1) in 100 microl of dimethylsulphoxide) daily. Body weights were determined weekly. The rabbits were then held until fully mature (6 months of age). Semen was collected and evaluated from sexually mature males on a weekly schedule for 5 weeks to maximize sperm output. An automated solid phase extraction procedure for monitoring exposures through isolation and quantification of Vin and its metabolic products was developed. Increased plasma levels of Vin and M2 were found throughout the experimental period. The exposed rabbits had a smaller weight gain during pubertal growth (approaching significance; P=0.059). At maturity, the accessory sex glands of the exposed animals weighed less than those of the controls (P=0.016). Surprisingly, the pooled sperm count of the exposed animals was significantly higher (P=0.017) than that of the unexposed animals. The anti-androgenic effects of Vin may have blocked the negative feedback mechanism of testosterone on the hypothalamus or pituitary gland, allowing for an increase in gonadotrophin release, and consequently increasing sperm production at puberty.

Semen quality and hormone levels among radiofrequency heater operators.

Approximately 9,000,000 US workers are occupationally exposed to radiofrequency (RF) radiation; over 250,000 operate RF dielectric heaters. Our purpose was to determine whether male RF heater operators experience increased adverse reproductive effects reflected in reduced semen quality or altered hormone levels. We measured incident RF heater radiation exposures and RF-induced foot currents at four companies. For 12 male heater operators and a comparison group of 34 RF-unexposed men, we measured 33 parameters of semen quality and four serum hormones. Despite wide variation in individual exposure levels, near field strengths and induced foot currents did not exceed current standard levels and guidelines. We observed minor semen quality and hormonal differences between the groups, including a slightly higher mean follicle-stimulating hormone level for exposed operators (7.6 vs 5.8 mIU/mL). Further occupational studies of RF-exposed men may be warranted.

Chemical and thermal effects on the viability and motility of spermatozoa from the turtle epididymis.

The viability and motility of spermatozoa harvested from the epididymides of turtles were estimated to elucidate properties that might enable them to be stored over long periods of time. Spermatozoa from the painted turtle, Chrysemys picta, were analysed and compared with spermatozoa from two other turtles, Trachemys scripta and Sternotherus odoratus using the Cellsoft analysis system for videotaped images. Spermatozoa from C. picta and T. scripta, suspended in F-10 medium, showed low motility (3-6% motile) and motion velocities, whereas the motility of spermatozoa from S. odoratus was higher (40% motile). Spermatozoa from C. picta and S. odoratus, but not T. scripta, had higher motilities and motion velocities when incubated at 2 degrees C before analyses. C. picta spermatozoa were unresponsive to calcium concentrations ranging from 10(-8) to 10(-1) mol l(-1), potassium concentrations ranging from 0. 1 to 10 mmol l(-1), and to pH values in the range 5.9-8.4. Spermatozoa from C. picta were sensitive to hypo-osmotic media, and showed reduced motility at 25% of normal osmolarity and no motility at 10% of normal osmolarity. Distorted cells and missing flagellae were noted at 50% of normal osmolarity. C. picta spermatozoa were viable up to 40 days after harvest when incubated at 4 degrees C; during this time, both motility and motion velocity were increased in response to 0.5 mmol 3-isobutyl-1-methylxanthine l(-1). Spermatozoa from turtles have osmotic properties and resistance to changing chemical environments similar to spermatozoa from other vertebrates that have internal fertilization, and appear to be stable over long periods of time compared with spermatozoa from other vertebrate species.

Reproductive function in relation to duty assignments among military personnel.

As a follow-up to the pilot study of semen quality of soldiers with various military assignments a larger, more complete study was conducted. Soldiers were recruited at Fort Hood, Texas. Thirty-three men were exposed to radar as part of their duty assignment in the Signal Corps, 57 men were involved with firing the 155 mm howitzer (potential lead exposure), and 103 soldiers had neither lead nor radar exposure and served as the comparison control group. Both serum and urinary follicle-stimulating hormone and luteinizing hormone and serum, salivary, and urine testosterone levels were determined in all men. A complete semen analysis was conducted on each soldier. For statistical analysis, the primary study variables were: sperm concentration, sperm/ejaculate, semen volume, percent normal morphology, percent motile, percent viable (both vital stain and hypoosmotic swelling), curvilinear velocity, straight-line velocity, linearity, sperm head length, width, area, and perimeter. Variables were adjusted for significant confounders (e.g., abstinence, sample age, race). No statistical differences (P < 0.05) were observed in any measurement. While these results are in agreement with two previous studies assessing soldiers firing the 155-mm howitzer, they contradict our previous report indicating that radar exposure caused a significant decrease in sperm numbers. A possible explanation is that the radar exposure in this study was that used in Signal Corps operations while the men in the previous study were using different radar as part of military intelligence operations. The data presented here in men firing the 155-mm howitzer combined with the results from the previous studies confirms that there are no deficits in semen quality in these men. The contradiction between the results of the radar exposure studies indicates that more data are needed to evaluate the relationship of military radar and male reproductive health.

Male reproductive effects of lead, including species extrapolation for the rabbit model.

The effects of elevated blood lead on semen quality were evaluated in the rabbit model and compared to published effects in humans. Mature, male rabbits were given lead acetate by subcutaneous injection in the dose range of 0 to 3.85 mg/kg on a Monday-Wednesday-Friday basis. In each of eight treatment groups, a dosing regimen was developed to produce blood lead levels of 0, 20, 40, 50, 70, 80, 90, and 110 microg/dL. A 5-week pre-exposure period was followed by a 15-week exposure testing period allowing for response through six cycles of the seminiferous epithelium. Semen analyses revealed that increased blood lead levels were associated with adverse changes in the sperm count, ejaculate volume, percent motile sperm, swimming velocities, and morphology. Hormonal responses were minimal. Testicular pathology revealed a dose-dependent inhibition of spermiation. For six measures of semen quality, threshold estimates ranged from 16 to 24 microg/dL. Using the species extrapolation factor derived in this study, a rabbit dose would have to be divided by 1.56 to obtain the equivalent human dose for an equal percentage decrease in sperm concentration; however, rabbits are 3.75 more sensitive in terms of absolute decrease in sperm count for a given blood lead level.

Semen analysis of military personnel associated with military duty assignments.

A collaborative study between the U.S. Army Biomedical Research and Development Laboratory (USABRDL) and the National Institute for Occupational Safety and Health (NIOSH) was designed to assess fecundity of male artillery soldiers with potential exposures to airborne lead aerosols. Potential exposure assessment was based upon information provided in an interactive questionnaire. It became apparent from extensive questionnaire data that many soldiers in the initial control population had potentially experienced microwave exposure as radar equipment operators. As a result, a third group of soldiers without potential for lead or microwave exposures, but with similar environmental conditions, was selected as a comparison population. Blood hormone levels and semen analyses were conducted on artillerymen (n = 30), radar equipment operators (n = 20), and the comparison group (n = 31). Analysis of the questionnaire information revealed that concern about fertility problems motivated participation of some soldiers with potential artillery or microwave exposures. Although small study population size and the confounding variable of perceived infertility limit the reliability of the study, several statistically significant findings were identified. Artillerymen who perceived a possible fertility concern demonstrated lower sperm counts/ejaculate (P = 0.067) and lower sperm/mL (P = 0.014) than the comparison group. The group of men with potential microwave exposures demonstrated lower sperm counts/mL (P = 0.009) and sperm/ejaculate (P = 0.027) than the comparison group. Variables used to assess endocrine, accessory sex gland, and sperm cell function were not different than the comparison group. Additional studies, incorporating larger numbers of individuals, should be performed in order to more optimally characterize potential lead and microwave exposure effects on male fecundity.

Measuring male reproductive hormones for occupational field studies.

As part of our longitudinal study of unexposed workers, we drew blood samples and analyzed the individual endocrine profiles for 45 men. The blood collection was between 8 AM and 8 PM, and three blood samples were drawn 20 minutes apart on three occasions during the course of the study (June, October, and February). Serum concentrations of follicle-stimulating hormone, luteinizing hormone, testosterone, and prolactin were determined. A component of variance model was used to estimate variability between the 20-minute blood draws. Statistical power analysis using this component showed that three blood draws provide a marginal improvement over a single blood draw in detecting population shifts. Also, if the prospect of three blood draws reduces subject participation by 10 to 20%, the increase in power would be negated.

Laboratory methods for assessing human semen in epidemiologic studies: a consensus report.

It is clear that additional methodologic work needs to be performed. Some data gaps described above are being actively investigated. Other standards were not addressed at this meeting; statistical handling of the data, differences among CASA machines, and factors to consider as potential confounders in analysis are just a few. These may be the subject of future workshops, which will also review progress made in the existing knowledge base. For now, this effort represents a first attempt to share information and to use it to encourage investigators in different laboratories to employ similar methods. In this way more direct comparisons among studies can be made, and our collective data base can be strengthened.

The effects of ethylene dibromide on semen quality and fertility in the rabbit: evaluation of a model for human seminal characteristics.

Mature (12 months old) male New Zealand White rabbits (8-10/group) were dosed subcutaneously with ethylene dibromide (EDB) in corn oil (untreated and vehicle controls, 15, 30, or 45 mg/kg body wt/day for 5 days). Weekly semen samples (for 6 weeks preexposure, during treatment, and 12 weeks postdosing [pd]) were analyzed for sperm concentration, number, morphology, viability, and motion parameters (velocity, linearity, beat cross-frequency, amplitude of lateral head displacement (ALH), and circularity), and semen pH, osmolality, volume, fructose, citric acid, carnitine, protein, and acid phosphatase (AP). Male fertility was assessed preexposure and at 4 and 12 weeks pd by artificial insemination of three females/male/time point with one million motile sperm. The percentage pregnant females, litter size, fetal body weights, and structural development were assessed. In the 45 mg/kg dose group of males there was 30% mortality and liver damage in 43% of the survivors as evidenced by increased levels of serum enzymes. Also in this group, EDB produced significant decreases in sperm velocity, percentage motility, and ALH (up to 25% at various times pd). There were also dose-related decreases in semen pH (up to 2%) and total ejaculate volume (up to 23%, 15 and 30 mg/kg dose groups only). AP activities were significantly elevated (up to 116%) 2 weeks pd in the 45 mg/kg dose group. All other semen parameters evaluated were unaffected. Male fertility and fetal structural development were also unaffected. Of the seven semen parameters perturbed by EDB in humans (Schrader et al., 1988), four were also affected in the rabbit (sperm velocity, percentage motility, pH, and volume), whereas sperm number, viability, and morphology were not. Thus, some of the male reproductive effects of EDB in the human have been modelled in the rabbit, although the rabbit appears not to be as sensitive, since semen parameters were affected only at doses close to the LD50 (55 mg/kg). The present study (together with other published data) suggests that the rabbit appears to be a potential model for male reproductive toxicity in humans, warranting further evaluation.

Longitudinal study of semen quality of unexposed workers. Sperm motility characteristics.

As part of a longitudinal study of human semen characteristics of unexposed workers, sperm motility measurements were made using computer-assisted sperm analysis. Motility analyses were conducted on monthly samples from 46 men for 9 months. Measurements of curvilinear velocity, straight-line velocity (VSL), linearity, amplitude of lateral head displacement (ALH), and beat-cross frequency were collected in eight microscope fields for each semen sample. The variability within a sample, between samples from the same individual (between monthly samples), and between individuals were calculated using a nested analysis of variance. For all sperm motility measurements, at least 90% of the variation was observed between cells within a semen sample. For all variables, the component of variation between subjects was the smallest percentage (ranging from 1.3% for ALH to 4.0% for VSL). When sample means were used in the nested analysis of variation, at least 75% of the variation was observed between samples from the same individual. These results will be useful in power calculations for future studies.

Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay.

Eight monthly semen samples from 45 men not known to be exposed to industrial toxicants were measured by the flow cytometric sperm chromatin structure assay (SCSA). This assay determines susceptibility of sperm DNA to in situ, acid-induced denaturation and is quantitated by the metachromatic shift of acridine orange fluorescence from green (native DNA) to red (denatured DNA). The observed green versus red fluorescence scattergram (cytogram) patterns were generally unique between donors and homogeneous within a donor over time. Within a donor, the cytogram patterns were the same whether intact sperm cells or detached nuclei were measured. For some individuals the cytogram patterns differed for some months and then returned to the original pattern. Intraclass correlations for mean and standard deviation of alpha t [alpha t = red/(red + green) fluorescence] were higher (.67 to .90) than any classically measured semen variables, suggesting that SCSA results within an individual were more consistent than other measures. Furthermore, average within-donor CV of alpha t parameters expressed as a percent of any given individual's means was around 10%, which is significantly lower than those derived from common semen measures. The SCSA is an objective, technically sound, biologically stable, sensitive, and feasible measure of semen quality.

Semen analysis and fertility assessment in rabbits: statistical power and design considerations for toxicology studies.

Semen analysis is commonly used in evaluating human response to reproductive toxicants. Serial semen samples can be collected from rabbits and fertility assessed by artificial insemination, hence this species is potentially well suited for male reproductive toxicity studies that might be extrapolated to humans. However, the size and cost of rabbits often restricts the number of animals used, reducing the sensitivity of such studies. Therefore, it was of interest to optimize study design for semen analysis and fertility assessment in rabbits. Semen samples were collected weekly from sexually mature New Zealand white rabbits and a range of parameters was analyzed (Semen--pH, volume, osmolality; Sperm--number and concentration, morphology, viability, percentage motility, motion characteristics; Seminal plasma--fructose, citric acid, carnitine and protein concentrations, acid phosphatase activity). Male fertility was assessed by inseminating female rabbits with the minimum number of motile sperm required for normal fertility, determined to be one million. The within- and between-buck variabilities were determined for all parameters and used to calculate the statistical power of different study designs. The variability of sperm number and concentration was decreased when measured in four ejaculates collected within a short period of time rather than in a single ejaculate; this was not true of other endpoints measured. In addition, use of preexposure observations further increased the statistical power for all of the parameters. These data can be used to determine the optimum design for studies of male reproductive toxicity using rabbits, with particular regard to cost and the number of animals used.

Longitudinal study of semen quality of unexposed workers: sperm head morphometry.

As part of a longitudinal study of human semen characteristics of unexposed workers, sperm head measurements were made using image analysis (Image Technologies Corp., Deer Park, NY). Morphometry was conducted on monthly samples from 45 men for 9 months. Measurements of area, perimeter, length, width, the width-length ratio, and the oval factor (4 [pi] area/perimeter) of 100 sperm heads per sample were obtained. The variability within a sample, between monthly samples from the same individual, and between individuals were calculated for each parameter. Tolerance intervals for each parameter were calculated, and are expected to contain 75% of all individual measurements. Similar intervals were calculated for the means and coefficients of variation of a semen sample. The largest source of variability was attributed to individual cell variation within a semen sample.

Semen quality in workers exposed to 2-ethoxyethanol.

To evaluate whether long term exposure to 2-ethoxyethanol (2EE) may affect semen quality, a cross sectional study was conducted among men exposed to 2EE used as a binder slurry in a metal castings process. Full shift breathing zone exposures to 2EE ranged from non-detectable to 24 ppm (geometric mean 6.6 ppm). Because of the potential for substantial absorption of 2EE through skin exposure, urine measurements of the metabolite of 2EE, 2-ethoxyacetic acid (2EAA) were conducted, showing levels of 2EAA ranging from non-detectable to 163 mg 2EAA/g creatinine. Only 37 exposed men (50% participation) and 39 non-exposed comparison (26% participation) from elsewhere in the plant provided a sperm sample. A questionnaire to determine personal habits, and medical and work histories, and a physical examination of the urogenital tract were also administered. The average sperm count per ejaculate among the workers exposed to 2EE was significantly lower than that of the unexposed group (113 v 154 million sperm per ejaculate respectively; p = 0.05) after consideration of abstinence, sample age, subjects' age, tobacco, alcohol and caffeine use, urogenital disorders, fever, and other illnesses. The mean sperm concentrations of the exposed and unexposed groups did not significantly differ from each other (44 and 53 million/ml respectively). No effect of exposure to 2EE on semen volume, sperm viability, motility, velocity, and normal morphology or testicular volume was detected, although some differences in the proportion of abnormal sperm shapes were observed. These data suggest that there may be an effect of 2EE on sperm count among these workers, although the possibility that other factors may be affecting the semen quality in both exposed and unexposed men in this population or that the results reflect bias introduced by the low participation rates cannot be excluded.

Morphometric and volumetric comparisons of human spermatozoa.

Morphometric measures and volumes of spermatozoa were determined for 28 human ejaculates which were previously analyzed for semen volume, sperm concentration, morphology, motility, and fertility by in vitro fertilization procedures (IVF). Morphometric measurements of sperm heads were analyzed using a Zeiss Videoplan computer, while spermatozoan volume was determined with an Elzone particle analyzer. Though a strong relationship was anticipated, correlations between the volumetric data and different morphometric measures revealed poor, insignificant values. This lack of correspondence may be due to individual differences in the thickness of the spermatozoa within a sample. Twenty-two of the ejaculates used in this study were classified as fertile and six were infertile according to the IVF procedure results. Correlations between the morphometric measurements and the volume determinations in the fertile group were all positive. In contrast, those of the infertile group were all negative with one exception (width vs. volume).

Effects of exposure to ethylene glycol ethers on shipyard painters: II. Male reproduction.

To determine whether 2-ethoxyethanol (2-EE) and 2-methoxyethanol (2-ME) affected the reproductive potential of exposed men, we examined the semen of 73 painters and 40 controls who work in a large shipyard. An industrial hygiene survey was performed to characterize the work environment. The men supplied information on demographic characteristics, medical conditions, personal habits, and reproductive history; underwent a physical examination; and provided a semen sample. Semen samples were analyzed for pH, volume, turbidity, liquidity, viability by stain exclusion and hypo-osmotic stress, sperm density and count per ejaculate, motility using a videotape technique, morphology, and morphometry. Serum was analyzed for testosterone, FSH, and LH. The industrial hygiene survey revealed that the painters were exposed to 2-EE at a time-weighted average (TWA) of 0-80.5 mg/m3 with a mean of 9.9 mg/m3, and to 2-ME at a TWA of 0-17.7 mg/m3 with a mean of 2.6 mg/m3. Painters had an increased prevalence of oligospermia and azoospermia and an increased odds ratio for a lower sperm count per ejaculate, while smoking was controlled. This finding is consistent with prior animal studies and with one human study. Potential biases and confounding of the data are discussed.

Longitudinal study of semen quality of unexposed workers. I. Study overview.

A longitudinal study of 45 men was conducted evaluating the semen quality of monthly samples collected over 9 months. The statistical variation of sperm count, semen volume, percentage of motile sperm, sperm velocity, sperm morphology, and sperm viability, assessed by both the vital stain and the hypoosmotic swelling (HOS) assay, were each evaluated using intraclass correlations and coefficients of variation. Sperm count and semen volume had large intraclass correlations (62% and 60%, respectively), indicating that if a subject has a high count or volume he will tend to continue to have high counts or volumes. On the other hand, sperm velocity had an intraclass correlation of only 16% indicating that fluctuations within a subject were nearly as large as fluctuations from subject to subject. The remaining parameters had intraclass correlations ranging from 42% to 47%. Sperm count, percent motile sperm, and semen volume each had large coefficients of variation (both between and within subjects). These variables, especially count, had relatively poor precision. Sperm velocity, percent motile sperm, percent normal morphology, the HOS assay, and the vital stain assay had lower coefficients of variation, indicating greater precision.

The effects of ethylene dibromide on semen quality: a comparison of short-term and chronic exposure.

Two occupational field studies were conducted to determine the effects of ethylene dibromide (EDB) exposure on male reproductive potential. The first study was a longitudinal study of 10 EDB-exposed forestry employees and 6 unexposed men conducted in Colorado in the summer of 1983. The exposure time was approximately 6 weeks. The second study was a cross-sectional study of 46 EDB-exposed papaya workers and 43 unexposed men conducted in Hawaii in December 1983 in which the average term of employment was about 5 years. In the longitudinal study, sperm velocity decreased in all 10 exposed men and in only two unexposed men. Semen volume was also decreased in 9 of the 10 exposed men (there was no change in the other man); only two unexposed men had a decrease in their semen volume. The longer term EDB exposure resulted in decreases in sperm motility and viability, suggesting that the short term exposure may slow sperm velocity, but longer exposures cause immotility and cell death. An apparent decrease in semen volume that was observed in the longitudinal study was not statistically significant in the cross sectional study of workers having chronic exposure. However, a significantly higher semen pH was observed in the exposed men compared to the unexposed men in the cross-sectional study. The results from both studies suggest that the accessory sex glands may be affected by EDB exposure.