Steven M. Schrader

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 individuals 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.

Designing prospective cohort studies for assessing reproductive and developmental toxicity during sensitive windows of human reproduction and development--the LIFE Study.

The relationship between the environment and human fecundity and fertility remains virtually unstudied from a couple-based perspective in which longitudinal exposure data and biospecimens are captured across sensitive windows. In response, we completed the LIFE Study with methodology that intended to empirically evaluate a priori purported methodological challenges: implementation of population-based sampling frameworks suitable for recruiting couples planning pregnancy; obtaining environmental data across sensitive windows of reproduction and development; home-based biospecimen collection; and development of a data management system for hierarchical exposome data. We used two sampling frameworks (i.e., fish/wildlife licence registry and a direct marketing database) for 16 targeted counties with presumed environmental exposures to persistent organochlorine chemicals to recruit 501 couples planning pregnancies for prospective longitudinal follow-up while trying to conceive and throughout pregnancy. Enrolment rates varied from <1% of the targeted population (n = 424,423) to 42% of eligible couples who were successfully screened; 84% of the targeted population could not be reached, while 36% refused screening. Among enrolled couples, ∼ 85% completed daily journals while trying; 82% of pregnant women completed daily early pregnancy journals, and 80% completed monthly pregnancy journals. All couples provided baseline blood/urine samples; 94% of men provided one or more semen samples and 98% of women provided one or more saliva samples. Women successfully used urinary fertility monitors for identifying ovulation and home pregnancy test kits. Couples can be recruited for preconception cohorts and will comply with intensive data collection across sensitive windows. However, appropriately sized sampling frameworks are critical, given the small percentage of couples contacted found eligible and reportedly planning pregnancy at any point in time.

Methods of monitoring menstrual function in field studies : efficacy of methods

Efficacy of methods for monitoring female reproductive potential under field study conditions was evaluated. Women (n = 10) were recruited to participate for two menstrual cycles on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Luteinizing hormone (LH), estrone-3-glucuronide, and pregnanediol-3-glucuronide were measured in daily morning urine and normalized to creatinine concentrations. These urinary measures were parallel to serum LH, estradiol, and progesterone profiles. Based on these urinary measures, 6 of 19 cycles were judged to be atypical. Transvaginal ultrasonography provided insights into ovarian activity during the atypical cycles. Of 13 LH surges detected by radioimmunoassay, 7 were not detected by a semiquantitative dipstick (OvuSTICK), perhaps due to that methods sensitivity to loss of LH immunoactivity caused by sample freezing. While intervals from salivary and vaginal mucous electrical resistance signals to the LH surge during typical cycles were similar to those reported previously, they were not predictive of ovulatory status during atypical cycles. Fifty-three percent of the cycles were misclassified on the basis of the basal body temperature rise. Cervical mucous color, amount, and consistency were not predictive of ovulation under these study conditions. The results from these 19 menstrual cycles provide information about the efficacy of various methods for characterizing menstrual function under field study conditions. In this regard, urinary endocrine measures are the most informative or practical.

Methods for assessing rat sperm motility

Computer-assisted sperm analysis (CASA) systems are becoming more widely used. With this spread of technology come more data from toxicology studies, designed to determine if treatment with putative toxicants affects sperm motion parameters. While these CASA methods provide us with more ways to evaluate toxicity and thus perhaps increase our chances of successfully protecting human health, there is also a greater likelihood that different laboratories will use different methods of collecting data on sperm motility. Different systems used with different methods in different laboratories will inevitably generate data that are difficult to compare. In a prospective attempt to address this issue of comparability and limit the problems, a group of individuals using CASA systems to analyze rat sperm motility convened to discuss methodologic issues, share data, and try to reach a consensus about methods for performing these studies. This article shares those meetings and data in the hope that common methods will enhance interlaboratory comparisons.

ASSESSMENT OF REPRODUCTIVE DISORDERS AND BIRTH DEFECTS IN COMMUNITIES NEAR HAZARDOUS CHEMICAL SITES. III. GUIDELINES FOR FIELD STUDIES OF MALE REPRODUCTIVE DISORDERS

Exposures to environmental toxicants can have detrimental effects on several aspects of human male reproduction: fertility, sexual function, hormone status, and pregnancy/birth outcomes. However, no simple prescreening methods are available for reliably identifying potential hazards; questionnaires alone are relatively imprecise and inefficient in the absence of field data. Multidisciplinary field studies are required that include detailed exposure information, health and reproductive histories, physical examinations, semen analyses, and possibly, hormone analyses. Semen analysis is a critical component of field studies for evaluating two aspects of male reproduction: 1) changes in sperm or seminal content, which may be indicative of adverse effects on the male reproductive system with possible implications for fertility potential; and 2) defects in sperm DNA or chromosomes, which may be associated with subsequent changes in viability during embryonic development and health risks to the offspring. Semen analyses may be tiered: 1) initially, each semen study may include conventional semen assays (concentration, motility, and morphology) as well as specific biomarkers indicated by the health effect of concern in the study cohort: and 2) archived samples (i.e., frozen, videotaped, or smeared) may be utilized in later second-tier analyses to further characterize specific findings. Before initiating any field study, it is cost effective to critically evaluate the suitability of the cohort by confirming exposure and determining that there are adequate numbers of male participants in each exposure category. Such evaluations must be based on the statistical sensitivities of the specific tissue biomarkers and health endpoints for detecting changes. This article summarizes the components of the ideal field study and identifies research needs for improving field studies of male effects and for understanding the mechanisms of male reproductive toxicity. Several promising semen methods currently under development are also discussed.

2,4-Dichlorophenoxyacetic acid residues in semen of Ontario farmers

Although paternal exposures to environmental toxicants probably play a role in adverse pregnancy outcomes, few data are available on the extent of this exposure. One semen and two 24-h urine samples were collected from 97 Ontario farmers who had recently used the phenoxy herbicides 2,4-D (2.4-dichlorophenoxyacetic acid) and/or MCPA ([4-chloro-2-methylphenoxyl acetic acid). Both samples were analyzed for 2,4-D using an immunoassay-based technique. Approximately 50% of the semen samples had detectable levels of 2, 4-D (> or =5.0 pph (ng/mL)). Semen levels of 2.4-D were correlated more closely with the second of the two urine samples. Although several studies have measured 2.4-D in the urine of applicators, this study is the first to attempt to measure 2,4-D levels in semen. As these pesticides can be excreted in the semen, they could be toxic to sperm cells and be transported to the woman and developing embryo/fetus. Further research is needed to understand how pesticide handling practices can affect semen pesticide residues and the relationship between the levels observed and reproductive health.

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.

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.

Sperm Viability: A Comparison of Analytical Methods

Summary:  Two methods of measuring human sperm viability, the stain exclusion assay and the hypoosmotic swelling (HOS) test, were evaluated. Human sperm were pretreated with 2.0% glutaraldehyde or 0.1% Triton X‐100 and compared to untreated controls. Approximately one half of the sperm were found to be viable in the control samples and nearly all sperm were non‐viable in the Triton X‐100 treated samples by both the stain exclusion and HOS assays. After glutaraldehyde pretreatment, presumably inactivating the spermatozoa, the HOS test revealed that most sperm were not viable, while the stain exclusion test found no difference between glutaraldehyde pretreated sperm and control sperm. Investigations with scanning and transmission electron microscopy demonstrated that the HOS test caused the membrane of the sperm tail to swell and the tail fibers to coil several times within the swollen membrane. It is concluded that the stain exclusion assay merely measures the structural integrity of the sperm membrane, whereas the HOS test also provides an indication of the physiological integrity of the sperm membrane.

The relation of computer-based measures of sperm morphology and motility to male infertility.

We investigated the relation between various sperm characteristics, including morphometric parameters, and impaired fertility among 596 men who participated in a national study. Semen was collected and processed by using a standardized protocol, and sperm measurements were made using a computer- aided sperm analysis instrument. We defined infertility in two ways: (1) the inability to father a child after trying for a year or longer, and (2) the number of children fathered. We found that all measures of sperm motion were decreased among men with impaired fertility. After adjustment for the other motion parameters and various potential confounders, however, only the percentage of progressive cells was associated with infertility. One morphometric parameter, the mean length/width ratio, was. consistently associated with both measures of infertility, even after adjustment for potential covariates. This measure was also strongly associated with infertility among various subgroups defined by poor sperm concentration, motility, and morphology. The sperm length/ width ratio appears to be an important correlate of infertility in males. (Epidemiology 1992;3:239-246)