Serum Estradiol Levels Are Inversely Associated With C-Reactive Protein in Premenopausal Women but Not Postmenopausal Women: a cross–sectional study

Background Epidemiological studies investigating the link between serum estradiol levels and inflammatory markers have produced inconsistent or conflicting results. We hypothesized that serum estrogen plays differing roles in inflammation according to menopausal status. Thus, we aimed to investigate the association between serum estradiol and C-reactive protein (CRP) levels in women according to their menopausal status. Methods We examined the association between serum estradiol and high-sensitivity CRP levels based on the menopausal status of 151 premenopausal women aged 21–52 years and in 394 postmenopausal women aged 46–75 years who participated in a health examination program. A multiple linear regression analysis was conducted using CRP level as the dependent variable. Results Multiple linear regression analysis showed that serum estradiol levels were inversely associated with CRP levels in premenopausal women (β coefficient = -0.298, P = 0.001). However, this was not the case in postmenopausal women after adjusting for age, body mass index, cigarette smoking, mean arterial pressure, fasting plasma glucose, triglyceride, high-density lipoprotein-cholesterol, aspartate aminotransaminase, and alanine aminotransaminase levels. Serum estradiol levels were inversely associated with CRP in premenopausal women but not in postmenopausal women. Conclusion Our findings suggest that lower estrogenic activity may at least partly contribute to the pathogenesis of chronic inflammation, particularly in premenopausal women. Log-transformed CRP levels significantly correlated with BMI, SBP, log-transformed triglycerides, log-transformed AST, and log-transformed estradiol levels in

Background Epidemiological studies investigating the link between serum estradiol levels and inflammatory markers have produced inconsistent or conflicting results. We hypothesized that serum estrogen plays differing roles in inflammation according to menopausal status. Thus, we aimed to investigate the association between serum estradiol and C-reactive protein (CRP) levels in women according to their menopausal status.
Methods We examined the association between serum estradiol and high-sensitivity CRP levels based on the menopausal status of 151 premenopausal women aged 21-52 years and in 394 postmenopausal women aged 46-75 years who participated in a health examination program. A multiple linear regression analysis was conducted using CRP level as the dependent variable.
Results Multiple linear regression analysis showed that serum estradiol levels were inversely associated with CRP levels in premenopausal women (β coefficient = -0.298, P = 0.001). However, this was not the case in postmenopausal women after adjusting for age, body mass index, cigarette smoking, mean arterial pressure, fasting plasma glucose, triglyceride, high-density lipoproteincholesterol, aspartate aminotransaminase, and alanine aminotransaminase levels. Serum estradiol levels were inversely associated with CRP in premenopausal women but not in postmenopausal women.
Conclusion Our findings suggest that lower estrogenic activity may at least partly contribute to the pathogenesis of chronic inflammation, particularly in premenopausal women.

Background
The development of cardiovascular diseases (CVD) is caused by multifactorial interrelated mechanisms, among which chronic low-grade inflammation plays a key role [1]. In women, menopause is another important factor that contributes to CVD, in addition to traditional risk factors such as obesity, hypertension, type 2 diabetes, and dyslipidemia [2]. Postmenopausal women are more susceptible to increased weight gain and visceral fat accumulation accompanied by marked decreases in estrogen levels [3,4]. These changes influence lipid metabolism and the vascular endothelium [5,6], thereby increasing the risk of atherosclerotic CVD in postmenopausal women [7]. inflammatory markers. In contrast to the consistent inverse relationship between serum testosterone levels and inflammatory markers in men [8,9], epidemiological studies that investigated the association between serum estradiol levels and inflammatory markers in women, particularly when categorized by menopausal status, have shown inconsistent and even conflicting results. Some observational studies found positive associations in postmenopausal women [10,11] and inverse associations in premenopausal women [12,13], whereas others did not reveal significant relationships [14]. Although the reasons for these discrepancies between serum estradiol levels and inflammatory markers among women are not clear, the effects of endogenous estrogen on chronic low-grade inflammation may differ by menopausal status.
C-reactive protein (CRP) has traditionally been considered a non-specific marker of inflammation, but recent epidemiological evidence has highlighted the significance of high CRP levels in patients with CVD through cross-sectional and longitudinal studies [15,16]. We hypothesized that serum estrogen plays differing roles in inflammation according to menopausal status. Thus, we performed this study to investigate the association between serum estradiol and CRP levels in women according to their menopausal status.

Study participants
We reviewed the medical records of 689 women who underwent medical examinations at the Health Promotion Center of the Gangnam Severance Hospital in Seoul, Korea, between November 2013 and July 2015. The participants voluntarily visited the health promotion center for regular health assessments. Natural menopause was defined as 12 consecutive months with no menstrual periods.
We excluded participants who met at least one of the following criteria: currently taking oral contraceptive medication; undergoing exogenous estrogen replacement; a history of tamoxifen therapy, induced menopause such as bilateral oophorectomy, radiation or drug-induced menopause; history of ischemic heart disease, cerebrovascular disease, thyroid, respiratory, renal, liver, or rheumatologic disease; CRP ≥10 mg/L; missing data; or not having fasted for 12 h prior to testing. We also excluded women who reported intermittent menstruation over the previous year to rule out the possibility of polycystic ovarian syndrome or perimenopause. After these exclusions, 545 participants

Data collection
Each participant completed a questionnaire that inquired about lifestyle habits, menstrual and medical history; cigarette smoking, alcohol consumption, and physical activity were determined based on the responses. Cigarette smoking was defined as an individual who currently smokes.
Alcohol drinking was defined as alcohol consumption on two days or more per week. Regular exercise was defined as engaging in purposeful physical activity three or more times per week. Menstrual history was determined via responses to the following question: "Has there been no menstruation for one year or more?" with three response options: "Yes," "No, but there has been intermittent menstruation over the last year," and "No, there has been no menstruation." Medical examinations were performed by trained medical staff using a standardized procedure. Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, with participants in light indoor clothing without shoes. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters (kg/m 2 ). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using the patient's right arm with a standard mercury sphygmomanometer

Statistical analysis
Normal distribution was evaluated with determination of skewness using a Kolmogorov-Smirnov test; the triglyceride, AST, ALT, estradiol, and CRP levels had skewed distributions. The clinical characteristics of the cohort were compared according to menopausal status using independent twosample tests or Wilcoxon's rank-sum tests for continuous variables, according to distribution normality, and the chi-squared test for categorical variables. Continuous data are presented as the mean ± standard deviation or median (interquartile range), whereas categorical data are presented as frequencies. A Pearson correlation analysis was conducted to examine bivariate correlations between log-transformed CRP and clinical variables. A multiple linear regression analysis was conducted with the log-transformed CRP level as the dependent variable to examine the independent association between estrogen and CRP levels. All analyses were conducted using the SAS statistical software (version 9.4; SAS Institute Inc., Cary, NC, USA). All statistical tests were two-sided, and P < 0.05 was considered statistically significant. Table 1 shows the clinical and biochemical characteristics of the participants, including 151 premenopausal women and 394 postmenopausal women, by menopausal status. The mean age of the premenopausal women was 42.7 ± 6.7 years, whereas that of the postmenopausal women was 58.1 ± 6.7 years. The mean values of age, BMI, blood pressure, and fasting plasma glucose, as well as the median values of triglycerides, AST, ALT, and CRP, were significantly higher in postmenopausal women than those in premenopausal women, while the mean HDL cholesterol and median estradiol levels were significantly lower in postmenopausal women than those in premenopausal women. Table 2 presents the results of the Pearson's correlation analysis between log-transformed CRP levels and clinical variables. Log-transformed CRP levels significantly correlated with BMI, SBP, logtransformed triglycerides, log-transformed AST, and log-transformed estradiol levels in premenopausal women. Among postmenopausal women, log-transformed CRP levels were significantly correlated with age, BMI, SBP, DBP, MAP, fasting plasma glucose, log-transformed triglycerides, HDL cholesterol, log-transformed AST, and log-transformed ALT levels but were not correlated with log-transformed estradiol levels. Table 3 presents the results of multiple linear regression analysis that shows the independent relationship between estradiol and CRP levels. Log-transformed estradiol was inversely associated with log-transformed CRP only in premenopausal women (β coefficient = -0.298, P = 0.001) but not in postmenopausal women after adjusting for age, BMI, cigarette smoking, mean arterial pressure, fasting plasma glucose, log-transformed triglycerides, HDL-cholesterol, log-transformed AST, and logtransformed ALT levels.

Discussion
In this cross-sectional study, we found that serum estradiol levels were inversely and independently associated with CRP in premenopausal women after adjusting for potential confounding variables.
Conversely, there was no such inverse relationship among postmenopausal women. Our findings are consistent with results of previous studies demonstrating that estradiol levels are inversely associated with inflammatory markers in premenopausal women [12,13].
The results from previous studies on the association between estradiol levels and inflammatory markers in women were inconclusive. Using the BioCycle Study dataset, Gaskins et al. reported an inverse association between estradiol and CRP levels in 259 reproductive-aged women in the United States, which is consistent with the findings of our study [12]. In contrast to our findings, serum estradiol was positively associated with CRP levels in 513 Italian postmenopausal women investigated in the Invecchiare in Chianti (InCHIANTI) study [11]. Therefore, additional longitudinal studies have been warranted to establish the relationship between serum estradiol levels and CRP on the basis of menopausal status.
The mechanisms underlying the menopause-specific relationship between serum estradiol and CRP level remain unclear, but some hypotheses have been suggested. Estrogen has an inhibitory effect on inflammatory cytokine production and inflammatory cell migration in non-reproductive tissues [17,18], and estrogen receptors are highly expressed in vascular smooth muscle and endothelial cells throughout the human body [18,19]. Estrogen's anti-inflammatory effects are in part mediated by nitric oxide (NO) production and cytokine suppression [17], particularly as NO is a key vasodilator [20,21]. NO also has an anti-inflammatory role in the endothelium owing to its role as a scavenger of reactive oxygen species (ROS) and an inhibitor of leukocyte recruitment [22][23][24]. Moreover, estrogen has been found to decrease the levels of tumor necrosis factor-α, a major pro-inflammatory cytokine [25]. These cascades potentially inhibit the synthesis and release of platelet-activating factors and chemokines such as interleukin-6 and may also down-regulate leukocyte-recruiting adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and Eselectin [26][27][28]. Estrogen could also decrease the production of ROS in mitochondria, which reduces chronic low-grade inflammation [29,30]. However, these effects of estradiol may differ according to menopausal status. The lack of an inverse relationship between estradiol and CRP in postmenopausal women may be explained by the observation that 80% of circulating estradiol in such individuals originates from the aromatization from testosterone, especially in adipose tissue; aromatase, which is the enzyme that converts testosterone into estradiol, is stimulated by inflammatory cytokines and CRP [11,31].
There are several limitations that should be considered when interpreting our findings. First, this study had a cross-sectional design; therefore, the results should be interpreted while taking into account causal and temporal considerations. Additional longitudinal studies are required to establish causality between serum estradiol levels and CRP based on menopausal status. Second, because the study participants were confined to women who underwent routine health promotion screenings at a single hospital and may therefore have comprised a relatively healthier proportion of the community, our cohort may not be representative of the general population. Third, the estradiol concentration was measured by ECLIA and not by liquid chromatography-mass spectrometry, even though the latter method is considered the gold standard for measuring serum estradiol. However, the ECLIA method has several advantages such as a rapid turnaround time, high-throughput, and full automation.
Fourth, serum estradiol levels and CRP levels may fluctuate during the menstrual cycle [32], and we did not measure serum estradiol levels and CRP levels at a common point during the cycle in our premenopausal participants. Lastly, although CRP is currently considered a reliable biomarker of chronic inflammation [16], caution should be taken when assuming anti-or pro-inflammatory activity based only on this biomarker, especially as we did not directly quantify other serum inflammatory markers such as tumor necrosis factor -α, serum interleukin-6, and other cytokines to support our findings.
Despite these limitations, there were also several strengths to this study. We assessed the association between serum estradiol and CRP in premenopausal and postmenopausal women separately, which may provide valuable insight into the association between serum estradiol and CRP on the basis of menopausal status. Moreover, a wide range of confounding factors closely related to chronic inflammation, including BMI, smoking status, blood pressure, fasting plasma glucose, triglycerides, HDL-cholesterol, and hepatic enzymes, were considered when performing multiple linear regression analyses. Additionally, to determine the true nature of the relationship between estradiol and CRP in pre-and postmenopausal women, our study excluded participants with induced or secondary menopause as well as women who used oral contraceptives or who were undergoing estrogen replacement therapy. Induced menopause causes a sudden onset of obesity and metabolic disturbances followed by an abrupt decline in ovarian hormones, which could cause chronic inflammation [33,34].

Conclusions
Our data show that serum estradiol levels are inversely associated with CRP levels in premenopausal women but not in postmenopausal women. Our findings suggest that lower estrogenic activity may at least partly contribute to the pathogenesis of chronic inflammation, particularly in premenopausal women.

Consent for publication
All participants provided written consent for their anonymized data to be used in publications.

Competing Interests
The authors declare that they have no competing interests. Data are expressed as mean ± standard deviation, median (interquartile range) or percentage (%). P-values were calculated g an independent two sample t-test, Wilcoxon rank-sum test, or chi-squared test.
Alcohol drinking refers to consuming on two or more days per week. Regular exercise refers to activity three or more times p eek.