Semen Quality and Reproductive Health of Young Czech Men Exposed to Seasonal Air Pollution

Selevan, Sherry G., Borkovec, Libor, Slott, Valerie L., Zudova, Zdena, Rubes, Jiri, Evenson, Donald P., Perreault, Sally D., Environmental Health Perspectives

This study of male reproductive health in the Czech Republic resulted from community concern about potential adverse effects of air pollution. We compared young men (18 years of age) living in Teplice, a highly industrialized district with seasonally elevated levels of air pollution, to those from Prachatice, a rural district with relatively clean air. Surveys were scheduled for either late winter, after the season of higher air pollution, or at the end of summer, when pollution was low. Participation included a physical examination, donation of a semen sample, and completion of a questionnaire on health, personal habits, and exposure to solvents and metals through work or hobby. Analysis of data from 408 volunteers showed that the men from Teplice and Prachatice were similar in physical characteristics, personal habits, and work- or hobby-related exposures. Sixty-six percent (272) of these men donated a single semen sample for routine semen analysis, computer-aided sperm motion analysis, and sperm chromatin structure assay. The mean (median) sperm concentration and sperm count were 61.2 (44.0) million/mL semen and 113.3 (81.5) million, respectively, and were not associated with district of residence or period of elevated air pollution. However, periods of elevated air pollution in Teplice were significantly associated with decrements in other semen measures including proportionately fewer motile sperm, proportionately fewer sperm with normal morphology or normal head shape, and proportionately more sperm with abnormal chromatin. These results suggest that young men may experience alterations in sperm quality after exposure to periods of elevated air pollution, without changes in sperm numbers. Key words, air pollution, epidemiology, human, semen, sperm chromatin, sperm count, sperm morphology, sperm motility. Environ Health Perspect 108:887-894 (2000). [Online 2 August 2000]

http://ehpnet1.niehs.nih.gov/docs/2000/108p887-894selevan/abstract.html

Air pollution in the Czech Republic increased dramatically with the advent of industrialization in the 1950s, primarily the result of increasing use of brown coal (with high sulfur content) for both home heating and industry. Sulfur dioxide emissions in Czechoslovakia amounted to 0.9 million tons in the 1950s and increased to 3.5 million tons by 1985 (1). This increase was particularly pronounced in the mountainous region of Northern Bohemia, where coal comes from mammoth open-pit mines and is used to heat homes and generate power for local industry. During the 1980s, ambient [SO.sub.2] levels associated with high levels of particulate matter (PM) in the Teplice district of Northern Bohemia frequently exceeded U.S. and Czech air pollution standards (2,3) in winter, when the use of coal increases and thermal inversions favor retention of the air pollution in the valley (4).

The Teplice Program, an international research program, was initiated in 1991 in response to concerns over potential health effects of this pollution. This program sponsored cooperative research among the Czech Institute of Hygiene, the Czech Ministry of the Environment, and the U.S. Environmental Protection Agency to compare health status in Teplice district to that in Prachatice district (5). We chose Prachatice because of its relatively cleaner air. A critical component of this program was the establishment of air monitoring in both districts to measure aerosol and gas-phase air pollutants [PM, including volatile and semivolatile polycyclic aromatic hydrocarbons (PAHs) and toxic metals] as well as [SO.sub.2], nitrous oxides ([NO.sub.x]), and carbon monoxide on an ongoing basis. Monitoring confirmed that levels of these air pollutants were considerably higher in Teplice than in Prachatice, and were higher in the winter than during the rest of the year in both districts (5,6). The Teplice Program includes studies of a number of health outcomes, including respiratory and neurologic effects in children, biomonitoring of mutagens in adults, and reproductive health in pregnant women and young men (5).

Reproductive health studies were prompted by reports that rates of conception and incidence of congenital anomalies were affected by seasonal increases in air pollution (7). To examine the potential relationship between the season of elevated air pollution and male reproductive health, we surveyed young (18-year-old) men and evaluated their semen quality. Metabolites of the PAHs present in this industrial air pollution can form protein or DNA adducts in body tissues (8) and thus have the potential to damage germ cell DNA. PAHs also reportedly alter male reproductive function in test species (9,10), providing additional rationale for this study. Furthermore, metals such as lead and cadmium that are present in the particulate fraction of air pollution have been associated with decrements in human semen quality (11).

Methods

This project was a collaborative effort between Czech and U.S. scientists. The study protocol was reviewed and approved by the Institutional Review Board of the Regional Institute of Hygiene of Central Bohemia, Prague, Czech Republic.

Subject recruitment. All young men turning 18 in the two districts over the 6 months before sampling were sent a letter from their district Hygiene Station with an appointment for a physical examination. Appointments were scheduled within 1 week's time in either early fall (1993) or late winter (1993 and 1994) to allow comparisons between recent exposures to periods of either high (winter) or low (summer) pollution. When each man presented for his physical examination, the reproductive study was explained to him, including his right to decline to participate and/or donate a semen sample, and written informed consent was obtained from each participant. No financial incentive was provided for participation. A pilot study was conducted in fall 1992 to estimate participation levels, establish field methods for the laboratory measures, and field test the questionnaire. Because methods for recruiting the participants in the pilot study differed from those used in the main study, data from the pilot study are not included in this report. However, a preliminary summary report of the study findings included the pilot data (5).

Questionnaire, physical examination, and semen sampling. The Czech study team traveled to the Teplice and Prachatice District Institutes of Hygiene for each sampling cycle (5 days) with the necessary supplies and equipment. Data were collected by questionnaire, physical examination, and semen sampling. A structured questionnaire, customized for use in the Czech Republic, was given by two trained interviewers. The purpose of the questionnaire was to obtain information on health status; other exposures such as metals, solvents, or pesticides encountered through hobbies or work (for those men undertaking apprenticeships); lifestyle data; and reproductive history including the date of last semen emission. Questions on other factors that could affect semen quality (such as fever, medications, exposure to X rays, cigarette use, consumption of alcohol and caffeinated beverages, and use of briefs) covered the previous 3 months. The physical examination included a urogenital evaluation and determination of testis volume based on caliper measures of testis length and width.

A single semen sample was collected on site by masturbation and sperm were videotaped within 1 hr of collection for subsequent motility analysis. Routine semen analysis included semen volume, sperm concentration [determined by hemocytometer according to World Health Organization guidelines; WHO (12)], total number of sperm per sample, percentage of motile sperm, percentage of sperm with normal morphology (entire cell considered), and percentage with normal head morphology. For the morphology assessment, we evaluated 300 sperm per sample from air-dried Papanicolaou-stained preparations and classified as either normal or abnormal according to strict criteria (12). The remaining semen was aliquoted into small tubes or straws, snap frozen on dry ice, and archived at -70 [degrees] C.

Computer-aided sperm analysis (CASA). Within 1 hr of collection, an aliquot of semen was diluted at least 1:1 with Dulbecco's phosphate buffered saline to obtain a concentration suitable for CASA analysis (13), loaded into a 20-micron-deep chamber (Microcell, Fertility Technologies, Inc., Natick, MA), placed on a stage warmer set to 37 [degrees] C, and videotaped (10x negative phase contrast with green filter). Video images were evaluated using a Hamilton-Thorne Integrated visual optical system (HTM-IVOS) semen analyzer (version 10.6; Hamilton-Thorne Research, Inc., Beverly, MA). We analyzed each field for 30 frames at 60 frames/second with minimum track length set at 20 points and examined enough fields to obtain velocity measures on at least 100 motile sperm. For oligospermic samples with [is less than] 100 motile sperm on the tape, mean velocities are included only when [is greater than or equal] 25 motile sperm were analyzed. We visually determined the percentage of motile sperm from the videotapes after scoring at least 100 sperm per sample. Because of technical difficulties, videotapes were unavailable for 10 men evaluated on 1 day in the Teplice (the late winter 1993 group).

CASA outcomes include indicators of a) sperm progression: straight-line velocity (VSL), straightness (STR = VSL/VAP x 100, where VAP is the average velocity along a mathematically smoothed sperm path), and linearity (LIN = VSL/VCL x 100, where VCL is the curvilinear velocity); and b) vigor: curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF), as described in detail elsewhere (14). Some of these outcomes have been associated with fertility status (15-18) and have been affected by occupational exposures (19,20). We also calculated two composite outcomes: the total number of motile sperm per sample (sperm count x %motile) and the total number of progressive sperm per sample (total motile x an index of progressive motility defined as percent of motile sperm with VSL 25 microns/second or greater).

Sperm chromatin structure assay (SCSA). Archived semen was shipped to South Dakota State University (Brookings, SD) for analysis using the SCSA, a measure of sperm nuclear integrity (21-23). Briefly, thawed and diluted semen was incubated for 30 sec in acid (pH 1.2) to potentially denature nuclear DNA, then immediately stained with the metachromatic dye, acridine orange (AO). AO intercalated into native double stranded DNA fluoresces green; AO complexed with single stranded DNA fluoresces red. We used flow cytometry to detect green (515-530 nm band pass filter) and red (630 nm long pass filter) fluorescence in 5,000 individual sperm per sample. The presence of DNA denaturation in each cell was observed as a shift from green to red fluorescence and was quantitated by the expression "[[Alpha].sub.t]," defined as the ratio of red/(red + green) fluorescence. We used the "cells outside the main population" (COMP [[Alpha].sub.t]) variable, which represents the percentage of cells containing denatured DNA. Normal sperm chromatin is resistant to acid induced DNA denaturation and fluoresces green. Increased red fluorescence indicates abnormal chromatin packaging and/or DNA damage. High COMP [[Alpha].sub.t] values have been associated with spermatogenic disorders and infertility (21-28).

Air pollution data and exposure categories. Air pollution data were provided by I. Benes and R. Stevens from the air monitoring program of the Teplice Project (5,6). These data include particulate matter [is less than] 10 [micro]m in aerodynamic diameter ([PM.sub.10]) obtained using the versatile air pollution sampler [VAPS (5,6)], PM-total suspended particulates (TSP), [SO.sub.2], CO, and [NO.sub.x]. Because VAPS data were incomplete for earlier phases of the study, we present both VAPS and TSP data. The correlation between TSP and [PM.sub.10] was very high (r = 0.96, p = [is less than] 0.01, n = 171) for those days where both were available. [SO.sub.2] data (an indicator of coal-derived pollution) were more complete than PM data. [PM.sub.10] levels were significantly correlated with [SO.sub.2] (r = 0.81, p [is less than] 0.01, n = 274), with [NO.sub.x] (r = 0.58, p [is less than] 0.01, n = 274), and CO (r = 0.49, p [is less than] 0.01, n = 252).

The process of spermatogenesis involves a series of complex steps (stem cell replication, meiosis, and spermiogenesis) over approximately 74 days in humans (29,30). Epididymal transit time [estimated at 3-12 days (31)] and abstinence interval (controlled in the analysis) can add several weeks to the time before mature sperm are ejaculated. Thus an exposure period of approximately 90 days is generally accepted as being of sufficient duration for detecting effects on any stage of spermatogeneis when using semen measures as the biologic end points. Therefore, for purposes of estimating and categorizing exposures relevant to seasonal changes in air pollution, the air pollution data for the 90-day period preceding sampling were considered relevant. Examination of the mean levels of pollutants (Table 1) shows that they were uniformly low in the 90 days preceding the fall sampling periods in both districts and in the late winter sampling period in Prachatice in 1994, and somewhat higher preceding the late winter sampling in Prachatice in 1993. Because all mean values were well below both U.S. (3) and Czech standards (2), and individual values rarely, if ever, exceeded air …

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Publication information: Article title: Semen Quality and Reproductive Health of Young Czech Men Exposed to Seasonal Air Pollution. Contributors: Selevan, Sherry G. - Author, Borkovec, Libor - Author, Slott, Valerie L. - Author, Zudova, Zdena - Author, Rubes, Jiri - Author, Evenson, Donald P. - Author, Perreault, Sally D. - Author. Journal title: Environmental Health Perspectives. Volume: 108. Issue: 9 Publication date: September 2000. Page number: 887. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2000 Gale Group.

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