CRP was inversely associated with estradiol across the menstrual cycle and positively associated with luteal progesterone levels. These associations persisted after controlling for demographic (i.e., age, race, and body mass index) and time-dependent (i.e., total cholesterol and other reproductive hormones) confounders. These results support the hypothesis that endogenous estrogens may act as antiinflammatory agents. Additionally, these findings have important implications for research and clinical practice because of CRP’s predictive value for cardiovascular disease in premenopausal women. In our study, the cardiovascular disease risk categorization of women by CRP level varied significantly across the menstrual cycle, suggesting the importance of consideration of menstrual cycle variability in measurement of CRP in women of reproductive age.
The cyclic changes in CRP and association with estradiol are supported biologically. Generally speaking, the events of the menstrual cycle involve inflammatory-like mechanisms (33
). Thus, observing cyclic changes in an inflammatory biomarker, such as CRP, across the menstrual cycle is not unexpected. The antiinflammatory effect of estradiol on CRP is consistent with estradiol’s effect on inflammation in many tissue types, as estrogens have negative effects on inflammatory cell migration and inflammatory marker production in diverse nonreproductive, nonimmune tissues (8
). Estrogen receptors are highly expressed in endothelial and vascular smooth muscle cells throughout the human body (9
). One of estrogen’s main antiinflammatory roles is through generating nitric oxide, an important vasoprotective molecule (34
). Apart from being a potent vasodilator, nitric oxide exerts an antiinflammatory role on the endothelium, as manifested by decreased leukocyte recruitment and scavenging of reactive oxygen species (35
). Estrogen has also been shown to reduce levels of tumor necrosis factor-alpha, a major proinflammatory cyctokine (36
). This in turn reduces the synthesis and release of chemokines like interleukin-8 and platelet activating factor, as well as down regulating adhesion molecules such as intercellular adhesion molecule 1 and E-selectin, which leads to recruitment of leukocytes (the hallmark of inflammation) (37
). Finally, estrogen can act as an antiapoptotic agent on various cell types including endothelial cells by preventing the release of cytochrome c
from the mitochondria, thus reducing subsequent vascular inflammation (38
Our finding that progesterone is positively associated with CRP is difficult to reconcile with many observations of progesterone exerting overall antiinflammatory effects on the immune system. Although progesterone can promote the chemotaxis of neutrophils and increase production of some inflammatory mediators (interleukin-6 and leukemia inhibiting factor) by monocytes (39
), progesterone also decreases natural killer cell activity, macrophage tumor necrosis factor, and nitric oxide synthase production and inhibits T-cell development and activity (40
). Therefore, our results suggesting that increases in progesterone typical of normally cycling women have an overall inflammatory effect require further study.
The observed fluctuation in CRP across the menstrual cycle and its association with endogenous reproductive hormones are consistent with results from several studies (13
) and at odds with 2 studies (16
). In general, previous studies were limited by small sample size (<37 women) (13
), restricted follow-up time (1 cycle) (14
), and issues with serum collection timing (13
). In general, studies with several CRP measurements were able to detect cyclic changes in CRP levels across the menstrual cycle. In particular, 2 studies with 7–12 measurements across the cycle observed fluctuations in CRP across the cycle, with maximum values occurring during the menses and the early follicular phase (13
). Similar to our findings with estradiol, those of Wander et al. (13
) and Blum et al. (14
) observed that a 10-fold increase in estrogen across the cycle resulted in a 29% and a 41% decrease in CRP, respectively. Wander et al. (13
) further observed a 29% decrease in CRP per 10-fold increase in progesterone. Most studies that compared only 3 phases per cycle did not observe the same cyclic changes in CRP (16
). Although 1 study did find significant differences in CRP when comparing 3 phases (follicular, midcycle, and luteal), they were not in the same pattern that we observed (15
Our study included intensive monitoring of a large number of young, ethnically diverse women throughout 2 menstrual cycles. To our knowledge, this is the largest, most comprehensive study of CRP across the menstrual cycle to date. Having 8 clinic visits per cycle timed with fertility monitors significantly decreased the probability of misclassifying menstrual cycle phase. The use of the hs-CRP assay as opposed to the standard CRP assay allowed us to measure very small amounts of CRP in the blood (range, from 0.5 to 10 mg/L) that are not quantifiable with the standard methods (range, from 10 to 1,000 mg/L). Although the regular CRP test is more common for patients at risk for infections or chronic inflammatory diseases, the hs-CRP assay is required to assess potential risk for heart problems among seemingly healthy people. Thus, given the range of levels observed in this study, regular CRP methods would have been insufficient. We had a wide variety of information on participants’ characteristics that increased our ability to adjust for confounding. Finally, the prospective design and exclusion criteria of the study strengthen the ability to draw inference, having reduced the potential for bias from known risk factors for inflammation and hormonal abnormalities.
Although hs-CRP is currently considered a reliable biomarker of chronic inflammation (24
), caution should be taken to infer an antiinflammatory mechanism based solely on this biomarker, because we did not have direct measurements of other serum inflammatory markers to confirm our results (i.e., serum interleukin-6, tumor necrosis factor-alpha, and other cytokines). While our study included the use of fertility monitors to time visits and visits were realigned on the basis of post-hoc evaluation of serum luteinizing hormone peaks, misclassification could have been introduced through mistimed sample collection; however, various indicators of successfully timed visits were found to be unrelated to hormone or CRP concentrations, and thus any misclassification is likely to be nondifferential. We restricted our study sample to healthy, regularly menstruating women in order to exclude potential confounders by design, but such restrictions could also limit the generalizability of our findings to other populations.
In conclusion, we observed that CRP concentrations varied across the menstrual cycle, attaining the highest and most variable levels during menses. Furthermore, CRP was significantly inversely associated with endogenous estradiol and positively associated with luteal progesterone. Given that CRP is one of the most commonly used markers of acute-phase reaction in clinical settings and the strongest predictor of cardiovascular disease in young women, measurement of CRP in clinical settings and future research studies should be standardized to menstrual cycle phase. To date, this study provides the most comprehensive assessment of the interplay between endogenous hormones and CRP among women of reproductive age. More research is warranted to further clarify the role of endogenous hormones on other biomarkers of inflammation in premenopausal women.