Markers of oxidative stress, particularly F2-isoprostane concentrations, vary (showing a significant, positive association with estradiol and inverse associations with SHBG and FSH) throughout the menstrual cycle. These associations persisted after controlling for demographic (i.e., age, race, and age at menarche) and time-dependent confounders (i.e., serum γ-tocopherol and beta-carotene levels, total cholesterol, and homocysteine). The direction of the association was also consistent with TBARS, another measure of lipid peroxidation, and several other markers of estrogen exposure such as age at menarche and body mass index. The observed associations between endogenous hormones and F2-isoprostane add critical understanding to the interplay of oxidative stress and endogenous hormones in women of reproductive age. Furthermore, if F2-isoprostanes are a specific marker of oxidative stress, this calls into question the commonly held hypothesis that endogenous estradiol acts as an antioxidant, protecting premenopausal women from risk of chronic disease.
Studies on the associations between reproductive hormone levels, in particular estrogens, and rigorously validated biomarkers of oxidative stress such as F2
-isoprostanes, among premenopausal women, are sparse. The observed positive association between F2
-isoprostane and estradiol is in contradiction to findings from in vitro and animal studies. However, caution should be taken when inferences are made from these previous studies, as they often rely on estrogenic compounds with widely different biologic (and potentially antioxidant) activity and concentrations of hormones that are significantly higher than the physiologic ranges (8
). The findings from observational studies are too sparse to clarify the role of estrogens as pro- or anti-oxidants. Investigations among postmenopausal women cannot be used to infer the relation between physiologic levels of natural hormones and oxidative status. A prior study focusing on older premenopausal women found a positive association between estrone metabolites and F2
). Of noticeable difference to our study, the median age of their premenopausal group of women (50 years compared with 27 years in our study) could affect the range of estradiol and estrone concentrations and the collection of data only in the early follicular phase when estrogen levels are low (compared with our 8 measurements across the menstrual cycle). Despite the differences, the conclusions are consistent: Neither study supports the commonly held hypothesis that levels of endogenous estradiol or its estrone metabolites favorably modify oxidative stress by decreasing F2
The significant inverse association between SHBG and F2
-isoprostane levels is biologically plausible given that SHBG binds to both estradiol and testosterone, mediating their effects (29
). The antiandrogenic effects of SHBG have been hypothesized to partially explain previous observations that SHBG is inversely associated with risk of breast cancer (30
) and type 2 diabetes (31
). Its antiandrogenic effects could also partly explain its inverse association with oxidative stress in the present study. Another potential mediator is through insulin resistance, which has been shown to be negatively associated with SHBG levels and positively associated with oxidative stress levels (32
The inverse association between F2
-isoprostane levels and FSH was not unexpected on the basis of the current knowledge regarding the inhibitory effect of estrogen on gonadotropin-releasing hormone production in the hypothalamus (33
) and the inverse relation with body mass index and age. However, the significant association between FSH and F2
-isoprostanes remained strong, even after adjustment for these factors, which suggests that FSH could act independently on F2
-isoprostane levels, although more research is needed to identify the exact mechanism.
Our finding that F2
-isoprostane levels vary during the menstrual cycle in concordance with estradiol concentrations uncovers limitations of previous research on F2
-isoprostane levels and clinical outcomes in premenopausal women; study designs need to take into account hormone levels or cycle variability and standardize the timing of the specimen collection. This important source of variability, specific to women, could help to explain the inconsistencies of findings between genders in previous studies that have attempted to link markers of oxidation to a number of clinical outcomes. For example, the Coronary Artery Risk Development in Young Adults (CARDIA) Study evaluated the relation between plasma F2
-isoprostanes and coronary artery calcification (34
). The mean and standard deviation (SD) of F2
-isoprostanes was 140.4 (SD, 55.6) pmol/L in men (n
= 1,302) and 190 (SD, 108.9) pmol/L in women (n
= 1,548). Subsequently, the adjusted odds ratios for any calcification in men versus women were 1.19 (95% confidence interval: 1.01, 1.40) and 1.13 (95% confidence interval: 0.89, 1.44), respectively (34
). Although the point estimates are similar across genders, the variability in women is larger than that in men, which could have reduced the precision and possibly result in the differences and conclusions. As demonstrated in , the variability of F2
-isoprostanes differs significantly by phase of the menstrual cycle. Thus, a standardized sample collection should take place during the early luteal phase when variability is minimized.
This study had a number of important strengths including the intensive monitoring of a large number of young and ethnically diverse women throughout 2 menstrual cycles. No previous study of premenopausal women had multiple longitudinal measures of F2
-isoprostane concentrations and reproductive hormone levels throughout the menstrual cycle. Moreover, multiple clinic visits timed with fertility monitors were a significant improvement over previous studies. Advantages of F2
-isoprostanes include their stability in their biologic surroundings, their lack of generation by enzymatic processes, and their availability in the urine as well as plasma, providing a noninvasive source of specimen for clinical analysis (18
). The prospective design and exclusion criteria of the BioCycle Study strengthen the ability to draw inference, having reduced the potential for bias from known risk factors for menstrual/hormonal disorders. In addition, standardized assessment of a wide variety of participant characteristics increased the ability to adjust for confounding.
Our study did, however, have a few potential limitations. Although our study included the use of fertility monitors to time visits, bias could have been introduced through mistimed sample collection; however, various indicators of successfully timed visits were found to be unrelated to estrogen or F2
-isoprostane concentrations, and thus any misclassification is likely to be nondifferential. In addition, F2
-isoprostane samples were not run in duplicate because of cost, and therefore measurement error was not adjusted for in the analysis. Similarly, although F2
-isoprostanes are currently advocated as sensitive and specific biomarkers of oxidative stress, care should be taken to infer an oxidative stress mechanism based solely on one biomarker. F2
-isoprostanes are highly specific markers of lipid peroxidation, but their use as a biomarker has some significant drawbacks including their trace levels, complex and expensive sample preparation, and analysis, as well as the fact that they are only minor products generated from arachidonic acid peroxidation, which is, in turn, a relatively minor component of total biologic polyunsaturated fatty acids (17
). Furthermore, F2
-isoprostanes may not provide sufficient evidence in view of reported intra- and intersubject variability (21
) and the fact that F2
-isoprostanes have biologic activities outside their role as biomarkers of oxidative stress (37
It is possible to speculate that, unlike an association with oxidative stress, the association between F2
-isoprostanes and estradiol may be a reflection of increased eicosanoid and prostanoid metabolism during the menstrual cycle. It has been shown that eicosanoid and prostaglandin precursors derived from arachidonic acid accumulate in the endometrium toward the time of menstruation (38
). Also, phospholipase A2
activity in the human endometrium is related to the stage of the menstrual cycle and suggests that arachidonic acid release may be influenced by estrogen and progesterone (39
-isoprostanes are derived solely from free radical attack on arachidonic acid/eicosanoid intermediates, and upregulation of the eicosanoid pathways during the menstrual cycle could simply provide more eicosanoid substrate for free radical attack leading to increased F2
-isoprostanes. The fact that TBARS tends to mimic these findings may also be attributable to the fact that TBARS generation is closely correlated with arachidonic levels in experimental lipid peroxidation (40
). Despite the above possibilities, our findings clearly suggest an association between endogenous estrogens and oxidative stress biomarkers.
We observed that levels of endogenous estradiol and factors that are positively related to estrogen exposure in premenopausal women are positively associated with F2-isoprostane levels throughout the menstrual cycle. SHBG and FSH were also negatively associated with F2-isoprostane levels. Although the effects of reproductive hormones on levels of F2-isoprostanes were very small in magnitude and might not have clinical importance, these findings help us to understand the biologic mechanisms that occur in vivo between a marker of oxidative stress and hormones. Of interest to future research, because of the significant cyclic variation in F2-isoprostane concentrations that we observed, cycle phase should be carefully accounted for when measuring levels of F2-isoprostanes in future studies of premenopausal women. To date, this study provides the most comprehensive assessment of endogenous hormones and oxidative stress interplay in women of reproductive age. If F2-isoprostanes are a specific marker of oxidative stress, these findings question the hypothesis that endogenous estrogen serves as a means of cardioprotection by inhibiting oxidative stress formation in premenopausal women. More research is warranted to further clarify the role of endogenous hormones on other biomarkers of oxidative stress, including markers of protein and nucleic acid damage as well as other biomarkers of lipid peroxidation in premenopausal women.