- Top of page
- Materials and Methods
ABSTRACT: The general population is exposed to phthalates through consumer products, diet, and medical devices. The present study explored whether phthalates, reproductive toxins in laboratory animals, were associated with altered sperm movement characteristics in men. Two-hundred twenty subjects provided a semen sample for computer-aided sperm analysis (CASA) and a urine sample for measurement of phthalate monoesters, monoethyl (MEP), monobenzyl (MBzP), mono-n-butyl (MBP), mono-2-ethylhexyl (MEHP), and monomethyl (MMP). Three CASA parameters, straight-line velocity (VSL), curvilinear velocity (VCL), and linearity (LIN), were used as measures of sperm progression, sperm vigor, and swimming pattern, respectively. There were suggestive dose-response relationships (shown as the predicted change in mean sperm motion parameter for the second and third tertiles compared with the first tertile; P value for trend) for MBzP with VSL (−2.36 μm/s, −2.81 μm/s; P = .09) and VCL (−1.67 μm/s, −2.45 μm/s; P = .4). There were suggestive negative associations between MBP and VSL (−3.07 μm/s, −2.87 μm/s; P = .08) and VCL (−3.25 μm/s, −3.46 μm/s; P = .2), and between MEHP with VSL (−1.09 μm/s, −2.73 μm/s; P = .1) and VCL (−0.29 μm/s, −2.93 μm/s; P = .3). In contrast to the other phthalates, MEP was positively associated with VSL and VCL but negatively associated with LIN. No consistent relationship was found for MMP and any sperm motion parameter. Although we did not find statistically significant associations, trends between CASA parameters, sperm velocity, and forward progression, and increased urinary levels of MBP, MBzP, and MEHP warrant further follow-up.
Currently, scientific and public concern exists regarding whether several commonly used industrial chemicals, such as phthalates, are associated with male reproductive toxicity in humans. These concerns stem from studies showing that a large proportion of the general population in the United States is exposed to phthalates (Blount et al, 2000b; Centers for Disease Control and Prevention [CDC], 2003) as well as animal studies showing they are reproductive toxicants (Gangolli, 1982; Reel et al, 1984; Agarwal et al, 1985; Parmar et al, 1986; Akingbemi et al, 2001). Furthermore, although the data are not conclusive, several researchers have found declining sperm counts in several developed countries (Carlsen et al, 1992; Comhaire et al, 1996; Gyllenborg et al, 1999; Swan et al, 2000), and there is concern over whether industrial chemicals may be partly responsible (Sharpe and Skakkebaek, 1993).
In one of the first human studies to explore associations between environmental levels of phthalates and human semen parameters, we recently demonstrated a relationship between environmental levels of phthalates and traditional semen analysis parameters categorized by World Health Organization (1999) categories for count, motility, and morphology (Duty et al, 2003). Specifically, we found dose-response relations between tertiles of monobutyl phthalate (MBP) and sperm motility (OR per tertile: 1.0, 1.8, 3.0, P value for trend = .02) and sperm concentration (1.0, 1.4, 3.3, P value for trend = .07). There was also a dose-response relation between monobenzyl phthalate (MBzP) and sperm concentration. We also found limited evidence for an association between monomethyl phthalate (MMP) with poor sperm morphology. To further explore the relationship between phthalate exposure and sperm motility, we investigated in a larger data set whether computer-aided sperm analysis (CASA) motion parameters were associated with environmental exposure to phthalates. CASA parameters are not as easily interpretable as World Health Organization semen analysis categories but may offer additional insights into whether any particular aspect of sperm movement would be differentially affected.
Phthalates are used in many consumer products such as carpet backing, soaps, shampoos, paints, glues, hair-sprays, nail polishes, insect repellents (Agency for Toxic Substances and Disease Registry, 1999), cosmetics, perfumes (Blount et al, 2000b), and medical products (Nassberger et al, 1987). Di(2-ethylhexyl) phthalate (DEHP), one of the more commonly used phthalates, leaches from blood products, and from intravenous and dialysate bags and tubing made with polyvinyl chloride (Nassberger et al, 1987).
There is consistent toxicological evidence of adverse developmental and reproductive effects of DEHP (Reel et al, 1984), butyl benzyl phthalate (BBzP) (Agarwal et al, 1985), and di-n-butyl phthalate (DBP) (Wine et al, 1997). Fewer live pups per litter, decreased live pup weights, and degeneration of the epididymis and testes have been found in pups after intrauterine DBP exposure (Wine et al, 1997). The National Toxicological Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) Expert Panel recently concluded that there is a larger risk of DBP associated reproductive and developmental toxicity following gestation and lactation exposures as compared to adult exposures (NTP, 2000). This finding, together with the fact that animal studies are conducted with doses higher than exposures expected in the general population, prompted the NTP-CERHR Expert Panel to determine there was low concern for adverse reproductive effects in humans from adult exposure to DBP.
Currently, we know of only two other human studies on the possible relationship between phthalates and testicular function (Murature et al, 1987; Rozati et al, 2002). However, interpretation of these results is difficult because both measured the diesters in semen, and no information was provided to explain how contamination from diesters in laboratory equipment was avoided, and one study did not address potential confounders.
The use of CASA in the clinical andrology laboratory has become commonplace due to the speed of analysis and objectivity of measurements. However, technical problems exist for several reasons, including the dependence of parameter estimates on frame acquisition rate and the number of frames analyzed, as well as the variability in the depth of the counting chamber (European Society of Human Reproduction and Embryology, 1996; Kraemer et al, 1998). CASA generally underestimates sperm movement parameters in samples with sperm counts >100 million/mL and overestimates them in samples with low counts; however, CASA accurately measures samples with sperm counts ranging from 40 to 100 million/mL (Mortimer et al, 1995). Currently, no standard protocols exist for CASA operation, but there are recommendations from the European Society for Human Reproduction and Embryology Andrology Special Interest Group (European Society of Human Reproduction and Embryology, 1996) for optimizing the CASA instrument for the specific setting of its use. The predictive value of CASA parameters has been compared with conventional semen analysis in a donor insemination program; CASA was superior in predicting which ejaculate achieved pregnancy compared to the traditional semen analysis parameters (Macleod and Irvine, 1995).
Based on the findings from rodent toxicity studies and our previous human study on a small number of men, we hypothesized that MBP, MBzP, and possibly MEHP will be associated with sperm motion parameters, whereas MEP and MMP will not.
- Top of page
- Materials and Methods
In the present study, we explored both linear and nonlinear relationships between CASA parameters and phthalate metabolites. Multiple linear regression analyses, in which phthalates were used as both a continuous measure and categorized into both tertiles and quintiles, were performed. This allowed us to explore threshold dose responses and U-shaped responses (Calabrese and Baldwin, 2001). Although they were not statistically significant, we found an overall pattern of decline in CASA parameters VSL, VCL, and LIN for phthalate monoesters MBP, MBzP, and MEHP. The absence of consistent statistically significant dose-response relationships may reflect a lack of power due to the relatively small number of subjects. Toxicological studies, primarily in rats, consistently show that select phthalate monoesters, including MEHP and MBP, are male reproductive and developmental toxicants (Parmar et al, 1986; Srivastava et al, 1990; Wine et al, 1997).
In laboratory animals, several researchers have explored the relationship between CASA parameters and chemical exposures, including epichlorohydrin and its metabolite, alpha-chlorohydrin (Slott et al, 1990, 1997). Epichlorohydrin, after 4 hours of inhalational exposure, transiently decreased path velocity despite no significant change in the percentage of motile sperm (Slott et al, 1990). Similarly, alpha-chlorohydrin given to male hamsters for 4 days resulted in a significant dose-dependent decline in VCL, VAP, and VCLselect despite no change in the percentage of motile sperm. In addition, alpha-chlorohydrin exposure was associated with a nonlinear impairment in in vitro fertilizing ability, which exhibited a threshold-like response (Slott et al, 1997). These studies suggest that CASA parameters may serve as a more sensitive maker of reproductive toxicity than semen parameters (Perreault and Cancel, 2001). One mechanism whereby sperm motion may be impaired includes oxidative stress and the production of reactive oxygen species and subsequent lipid peroxidation of sperm plasma membrane (Aitken, 1997; Storey, 1997; Armstrong et al, 1999).
Although CASA parameters may prove to be sensitive biomarkers of reproductive toxicity in humans, they are difficult to compare across studies because of the use of different CASA instruments and settings (Davis et al, 1992). Despite this limitation, human studies have shown that CASA parameters can be used to predict fertility (Aitken et al, 1982) and pregnancy (Macleod and Irvine, 1995; Larsen et al, 2000). Furthermore, there are also epidemiologic studies using CASA parameters as a marker of altered semen quality. Selevan et al (2000) examined the association between air pollution levels and VSL, VCL, and LIN and found that medium levels of air pollution adversely affected VCL but improved LIN. High air pollution levels, however, improved VSL and VCL but unexpectedly decreased LIN. The inference is that although the sperm traveled faster, the pattern was more erratic, and therefore, forward progression actually decreased.
The strengths of the present study include the availability of a reliable biomarker of phthalate exposure instead of relying on self-reported exposures. Biomarkers have the potential to quantify exposures to chemicals from all routes of exposure, including oral, dermal, inhalation, and ingestion (Teass et al, 1998). Furthermore, by measuring the monoester phthalate (ie, the metabolite), we avoided difficulties resulting from contamination from plastic products, such as the urine specimen collection cup (Blount et al, 2000a). One limitation in the present study was that if phthalates are associated with a complete lack of sperm motility, this study is not able to detect this, because by definition, we were able to explore motion parameters only on motile sperm.
Evidence of widespread exposure of the general population to phthalates comes from a recent study on phthalate metabolite levels in urine collected for the Second National Report on Human Exposure to Environmental Chemicals, NHANES 1999–2000 (CDC, 2003). The NHANES survey collected biological samples and information about the health and diet of people in the United States (National Center for Health Statistics, 2001). Four phthalate metabolites, MEP, MEHP, MBP, and MBzP were present in more than 75% of US subjects sampled (CDC, 2003). In the present study, 100% of subjects had measurable MEP, 95% had detectable MBP and MBzP, whereas 75% had detectable MMP and MEHP levels.
The NHANES 1999–2000 survey contains data on monoester levels stratified by gender or age (6–11 years, 12–19 years, and ≥20 years). The NHANES report did not include cross-stratification by age and gender (eg, the report did not present data for a substratum consisting entirely of adult males age ≥20 years). Therefore, the NHANES male stratum included men from 6 to >60 years of age, whereas the age strata included both men and women. This lack of cross-stratification by age and gender makes the NHANES male stratum not entirely comparable to the present study on adult men because of trends in phthalate monoesters with age. For instance, children (6–11 years) and adolescents (12–19 years) had higher median levels of MBP, MBzP, and MEHP than adults (≥20 years). In contrast, median levels of MEP were lower for the age group 6–11 years than for the other two age groups. It is unknown whether age and gender differences in monoester concentrations reflect differences in exposure, body-size relationships, or metabolism. Because of the absence of a stratum of adult men in the NHANES report, the comparisons made between the present study and NHANES are meant as qualitative guidance but should not be used to determine precise quantitative differences. The samples in the present study and the NHANES samples were both analyzed by the same CDC laboratory.
In the present study, unadjusted median MEP levels (152.7 ng/mL) were similar to median levels in NHANES men (154 ng/mL), whereas MEHP was higher in the present study (6.1 ng/mL) as compared to NHANES (3.40 ng/mL). In contrast, in the present study, median MBP (17.8 ng/mL) and MBzP (9.9 ng/mL) levels were lower than in the NHANES data set (23.1 ng/mL and 17.7 ng/mL, respectively). The inclusion in the NHANES male stratum of children and adolescents with higher MBP and MBzP levels than adults may account for the higher median levels of MBP and MBzP in NHANES as compared to the present study. In contrast, the high levels of MEHP found in the present study would be even higher than NHANES levels if children and adolescents, with higher MEHP levels than adults, were excluded from the NHANES male stratum. MMP was not measured in the NHANES data set.
There is controversy about the best way to correct for urine volume when using a single spot urine sample (Boeniger et al, 1993; Teass et al, 1998). In our sensitivity analysis, we used specific gravity criteria to exclude 33 samples and analyzed the data without adjusting the remaining phthalate levels. The results were consistent with the primary analysis, although the relationship between MBP and MBzP became weaker, while the relationship for MEHP and both VSL became stronger. This may indicate that the additional measurement error introduced by not using specific gravity adjustment biased the results toward the null hypothesis, making relationships more difficult to detect.
It is interesting to note that study subjects were exposed to several phthalates simultaneously, which raises the issue of how to explore relationships between semen parameters and exposures to multiple phthalates that may act via similar mechanisms. Gray and colleagues (2000) suggested that risk assessments for phthalate-induced reproductive toxicity should consider phthalates as a group and include exposures from multiple sources (Gray et al, 2000). Although they provide preliminary phthalate ester toxic equivalency factors (PE-TEFs) for reproductive toxicity induced in utero, they do so to stimulate discussion and further research about how we should estimate cumulative and aggregate risk to phthalates. Additional dose-response studies are necessary before we can apply PE-TEFs in both toxicological and epidemiological studies.
In conclusion, although we did not find statistically significant associations between CASA parameters and adult exposure to phthalates, there were trends that warrant further follow-up. These data extend the results of our previous study (Duty et al, 2003) that found an association between MBP and lower sperm motility. Although intriguing, these results are preliminary and should be explored in larger, as well as different study populations.