In the present study, urinary MEHP concentrations were inversely associated with circulating testosterone and estradiol levels in adult men recruited through an infertility clinic. We also found evidence for inverse associations between MEHP, MEHHP, MEOHP, sum of measured DEHP metabolites and FAI, and a positive association between MEHP% and T:E2
ratio. We previously reported associations between MBP and inhibin B, and between MBzP and FSH, among a smaller and overlapping group of men in our study (Duty et al. 2005
). However, in the present analysis we observed no relationships among MEP, MBP or MBzP with any of the measured hormones.
Only a limited number of studies have investigated the relationship between human phthalate exposure and circulating reproductive hormone levels or hormone indicators (Pan et al. 2006
; Jonsson et al. 2005
; Main et al. 2006
; Swan et al. 2005
), and only two other previous studies have been conducted among men (Pan et al. 2006
; Jonsson et al. 2005
). Pan et al. (2006)
studied 74 workers in a Chinese factory exposed to high levels of DEHP and DBP in the production of unfoamed PVC, and 64 unexposed workers. The authors reported inverse associations between urinary MBP and MEHP concentrations and free testosterone among the workers, but no relationship between the phthalate metabolites and estradiol. Interestingly, although MEHP concentrations in the present study among non-occupationally exposed men were several orders of magnitude lower than those measured in the Chinese workers with occupational exposure (Pan et al. 2006
), the evidence for decreased testosterone in relation to DEHP and/or MEHP was consistent between the two studies.
Conversely, the findings in the present study were inconsistent with those reported in the other study of non-occupationally exposed Swedish men by Jonsson and colleagues (2005)
. In that study no associations between urinary MEHP and testosterone or estradiol were observed. It is possible that the discrepancies in findings between studies may be explained by differences in study design or laboratory methods. For example, there were large differences in the ages of the study populations and the methods of recruitment. The Swedish study population consisted of young men (median age 18 years, range 18-21 years) undergoing a medical examination before military service. Since approximately 95 percent of young men in Sweden undergo the conscript examination, these young men reflected the general population of young Swedish males. In contrast, in the present study the median age was 35.5 years and ranged from 22 to 54 years. However, it is unclear whether men presenting to an infertility clinic are more ‘susceptible’ to exposure to reproductive toxicants than men from the general population or whether older men are more ‘susceptible’ to reproductive toxicants because of an age related response. Other differences between the two studies include major differences in participation rates (14% in the Swedish study and 65% in the US study) and differences in the analytical methods used to measure urinary phthalate metabolites, where the method in the present study was more sensitive than that used in the Swedish study.
Consistent with our findings, animal and in vitro
studies have demonstrated that several phthalates, including DEHP or its metabolites, are endocrine disrupting chemicals that possess anti-androgenic activity and reduce testosterone and estradiol levels (ATSDR 2002
). The mechanisms by which these alterations are occurring are not fully established. Recent evidence suggests phthalates may inhibit expression of genes or proteins related to steroidogenesis, such as steroidogenic acute regulated protein (StAR), peripheral benzodiazepine receptor (PBR), P450 side chain cleavage (P450scc), and peroxisome proliferator activated receptors (PPAR) in Leydig cells (Borch et al. 2006
). However, this and most other studies of DEHP or MEHP anti-androgenic activity that have been conducted to date were performed in rat fetal testis and thus may not reflect what occurs in adult humans. Additional research is needed on reproductive health effects from phthalate exposures in adulthood, and the involvement of alternative mechanisms such as hepatic SHBG transcription activity and altered pituitary function should be considered.
We found limited evidence for interaction by MEHP% of the inverse association between MEHP and FAI. In multiple logistic models for having FAI in the lowest quartile, the association was much stronger among men with high MEHP%. This was consistent with our a priori hypothesis that a high percentage of MEHP (a bioactive hydrolytic metabolite of DEHP) to the sum of all measured DEHP metabolites reflects increased susceptibility to DEHP exposure due to less efficient oxidation and excretion of DEHP and/or MEHP. However, we did not find evidence for modification of the other observed associations by MEHP%, though this may be due to the small number of men with oxidized DEHP metabolites measured (n=221). The possibility of effect modification by MEHP% should be tested in future epidemiological studies with larger sample sizes.
Another secondary finding from the present study was a suggestive positive association between T:E2
ratio and MEHP, and a strong positive association between T:E2
ratio and MEHP%. A larger T:E2
ratio is a marker for reduced aromatase activity. Consistent with these findings, animal and in vitro
studies have demonstrated that DEHP and/or MEHP can suppress aromatase in a human adrenocortical carcinoma cell line, in cultured rat ovarian granulosa cells, and in the brain and testes of young male rats (Noda et al. 2007
; Andrade et al. 2006
; Kim et al. 2003
; Lovekamp and Davis 2001
; Davis et al. 1994
). Although suggested as a potential treatment for certain cases of male infertility (Liu and Handelsman 2003
; Schiff et al. 2007
), aromatase suppression could be associated with adverse effects on male reproduction (Carreau et al. 2006
). A potential alternative explanation for an increased T:E2
ratio in relation to DEHP and/or its metabolites in the present study could be through an increase in estradiol metabolism through PPARα-dependent mechanisms (Corton et al. 1997
Recent experimental evidence has suggested that estradiol plays an important role in spermatogenesis and male reproduction, and that estradiol is a potent inhibitor of male germ cell death (Pentikainen et al. 2000
; Hess et al. 1997
). We also recently reported strong inverse associations between estradiol levels and human sperm DNA damage (Meeker et al. 2008
). Thus, it is possible that a decrease in estradiol levels and/or aromatase activity may explain our previous observation of a significant increase in sperm DNA damage associated with increased urinary MEHP when accounting for oxidized DEHP metabolites (Hauser et al. 2007
). Alternatively, the significant positive association between MEHP% and T:E2
ratio may reflect relationships between aromatase and enzymes involved in efficient DEHP or MEHP metabolism. Future research is needed to help explain these findings.
A potential limitation of the present study is the measurement of urinary phthalate metabolites and serum hormones at a single point in time. Phthalates are rapidly metabolized and excreted, and metabolite concentrations in urine only reflect exposure in the preceding 1 or 2 days. Several studies have explored temporal variability of urinary phthalate metabolites, where high within-individual variability has been reported over the course of several days to months (Fromme et al. 2007
; Hoppin et al. 2002
; Teitelbaum et al. 2008
). Nevertheless, we demonstrated that a single sample may adequately predict average monoester concentrations over a 3-month period in adult men (Hauser et al. 2004
). Serum hormone levels may also vary within an individual over time, but a single measure has been shown to provide a reliable measure in population studies (Bjornerem et al. 2006
; Schrader et al. 1993
; Vermeulen and Verdonck 1992
; Bain et al. 1988
). In addition, requiring multiple blood samples from participants may result in a reduced participation rate and lower statistical power.
Concentrations of (unadjusted) urinary phthalate metabolites measured in the present study were compared to those among U.S. males measured as part of the 2003-2004 National Health and Nutrition Examination Survey (NHANES) (CDC 2008
). Metabolite distributions were generally similar between the present study and the national data, although in the present study concentrations of MEP, MBP and MBzP were slightly lower, and concentrations of MEHP, MEHHP and MEOHP were somewhat higher. For example, the median and 95th
percentile values for MEHP (unadjusted for specific gravity) in the present study were 6.0 and 112 ng/ml, respectively, compared to 2.2 and 33.3 ng/ml in males from NHANES 2003-2004.
In conclusion, the present study found that urinary DEHP metabolites, at levels that are representative of those found among the general US population, may be associated with altered steroid hormone levels and perhaps aromatase activity. Additional work is needed to confirm these findings and determine clinical implications of subclinical alterations in hormone levels in adult men following exposure to environmental EDCs.