Moderate increases in serum C-reactive protein (CRP) and homocysteine have been associated with higher risk of cardiovascular disease in several observational studies.1,2 Research has shown that use of hormones modulates the levels of CRP and homocysteine in women. However, most of these investigations were performed in women aged 45 years or older.2–5 The effect of oral contraceptives (OCs) on these two biomarkers has not been adequately explored.
Third-generation OCs (containing desogestrel or gestodene) were introduced to reduce severe adverse effects of OC use, especially, cardiovascular diseases.6–9 However, evidence suggests that these preparations do not reduce, but may even increase, the risk of venous thromboembolism compared with previous generations.8,10,11 In addition, the recent observation that hormonal contraception use is associated with more rapid human immunodeficiency virus (HIV) disease progression, suggests that there is an urgent need to better explore the immune inflammatory effects of OCs.12,13
C-reactive protein is a well-known acute phase response factor. Recently, high-sensitivity CRP assays were developed with detection limits of approximately 0.1 mg/L,14,15 for the assessment of low-grade chronic inflammation, which is implicated in cardiovascular disease development. Analyses for vascular risk stratification generally used high-sensitivity CRP thresholds of less than 1.0 mg/L, 1.0 to 3.0 mg/L, and 3.0 mg/L or more.16 More recently, high-sensitivity CRP levels were further categorized as less than 0.5 mg/L (protective cutoff), 0.5 to less than 1.0 mg/L (no risk level); 3.0 to less than 10.0 mg/L (high risk level), and 10.0 mg/L or more.1,17
Homocysteine is an amino acid that derives from demethylation of methionine. High levels of homocysteine are related to increased risk of venous thrombosis, cardiovascular diseases, and disorders of the central nervous system.4,5,18 Homocysteine metabolism may be influenced by dietary habits and lifestyle factors.5 In particular, homocysteine is inversely related to folate.5 Few studies investigate the hormonal modulation of homocysteine in premenopausal women,19–21 whereas several studies examined hormone therapy effects in postmenopausal women giving mixed results.2,5
The aim of this study was to compare high-sensitivity CRP and homocysteine levels in young healthy nonobese women who do and do not use third-generation OCs using the newly defined high-sensitivity CRP levels.
MATERIALS AND METHODS
Healthy white Italian women were recruited in Udine from May 2006 to May 2007. Consecutive enrollment was done through announcements at the University of Udine Campus. Women were not paid for participation in the study. Inclusion criteria were 18–30-year-old, premenopausal, nonpregnant, nonbreastfeeding women, without current infections, chronic inflammatory diseases, or major diseases such as diabetes, cardiovascular disease, or malignancies. Women were enrolled in the OC user group if they were using third-generation OCs only for at least 2 months. Women were considered eligible for inclusion in the non–OC user group if they had not used any hormonal treatment for at least 2 months before enrollment. Women who had recent viral or bacterial infections were excluded. All participants completed a self-administered questionnaire assessing demographic factors (age, ethnic group, and education), medical history (pregnancies, past or recurrent illness), contraceptive histories, health behaviors including alimentary habits, coffee and alcohol drinking, use of diet supplementation, smoking, and physical activity.22 Of 300 women screened, 282 participants fulfilled inclusion and exclusion criteria. Five women had incomplete data, leaving a total of 277 women for analyses.
This study complies with the Declaration of Helsinki and was approved by the local institute review board of Udine University, on May 2, 2006. Written informed consent was obtained from all subjects.
Blood samples were obtained from seated and fasting subjects in the morning from the antecubital vein using evacuated tubes (Vacutainer Tubes, Becton-Dickinson, Franklin Lakes, NJ) with ethylenediamine tetraacetic acid as anticoagulant, or without anticoagulant but containing beads for better serum separation. Samples were collected at the Clinical Analyses Laboratory, University of Udine, which serves as outpatient and hospital laboratory in Udine. Personnel executing the collection and measurement of samples were blinded to clinical, demographic, and habit data. Full blood counts and hemoglobin measurements were immediately performed on an automatic cell counter, Cell-Dyn Sapphire analyzer (Abbott Laboratories, Chicago, IL).
Ethylenediamine tetraacetic acid blood samples were immediately protected from light and centrifuged at 3,000 g for 15 minutes at 10°C, and the plasma was aliquoted and stored at –80°C. Serum was separated less than 30 minutes after blood coagulation by centrifugation at 2,200 g for 5 minutes at 15°C. Aliquots of serum samples were frozen at –80°C for further analysis.
Serum CRP was measured using particle-enhanced immunonephelometry (Behring Nephelometer Analyzer, BN II, Dade Behring, Marburg, Germany) by the highly sensitive method CardioPhase high-sensitivity CRP assay (Dade Behring), which was calibrated with an internal standard (Rheumatology Standard SL) traceable to International Certified Reference Material 470 according to Food and Drug Administration requirements. Detection limit of the assay was 0.15 mg/L or less. Samples with values below the detection limit were set at 0.15 mg/L for statistical calculations. The intraassay and interassay coefficients of variation (CVs)% were 2.6% and 6.5%, respectively.
Serum homocysteine concentrations were measured by a fluorescence polarization immunoassay (AxSYM Homocysteine, Abbott Diagnostics, Wiesbaden, Germany), with automated Abbott AxSYM system (Abbott Diagnostics). Analytic sensitivity of the assay was 0.8 micromole/L or less. Intraassay and interassay CVs% were 2.3% and 3.2%, respectively. Reference interval found by the manufacturer for the female population was 4.6–12 micromole/L. In addition to the manufacturer’s upper threshold, we used the 15 micromole/L or more cutpoint as high homocysteine level based on previous studies.5
Plasma folate concentrations (3.0–12.5 microgram/L reference interval) were determined using a chemiluminescent microparticle folate-binding protein immunoassay (Architect Folate, Abbott Diagnostics). Analytic sensitivity of the assay was 0.8 microgram/L or less. Intraassay and interassay CVs% were 3.0% and 5.4%, respectively.
Serum concentrations of total bilirubin, triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), creatinine, and glucose were measured on Modular analyzer (Roche Diagnostics, Mannheim, Germany) by appropriate reagents (Roche Diagnostics). Low-density lipoprotein (LDL) cholesterol was calculated from triglycerides, total, and HDL cholesterol according to the Friedewald equation.
The Kolmogorov-Smirnov test was used to assess the normality of data distribution. Normally distributed variables were presented as mean and standard deviation (±SD). For skewed markers, median (25th to 75th percentile interquartile range [IQR]) values were reported and nonparametric tests used. The t test or Mann-Whitney U test was used for comparison of continuous variables, as appropriate. The difference of proportions between OC users and non–OC users was assessed by χ2 test or Fisher exact test, as appropriate. Univariable odds ratios (ORs) and 95% confidence intervals (CIs) were evaluated for categorical variables. In addition, logistic regression was performed to evaluate the difference in high-sensitivity CRP between OC users and non–OC users after adjustment 1) for age, body mass index (BMI), smoking, and white blood cells (WBC) and 2) for age, BMI, smoking, WBC, cholesterol, triglycerides, albumin, bilirubin, ALP, and lymphocytes. Bivariate relationships were evaluated by Spearman Rho test (ρs). All tests were 2-tailed. P<.050 was considered statistically significant. At an alpha level of 0.05, we had 94.8% power to detect a difference in CRP proportions between the OC and the non-OC groups. After adjusting for the correlation among the variables included in the multivariable logistic regression model, we still had greater than 90% power to detect a difference in the groups. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL).
On average, the 277 study women were 23±4.6 year olds and had a BMI of 20.9±2.21 kg/m2; 97.5% were nulliparous, enrolled at or employed by Udine University; 75% had university-level education; all had a middle-class socioeconomic status; and 21% were smokers. A total of 77 study participants (77 of 277, 28%) were currently taking third-generation OCs. Of these, 24 (31%) were formulations containing desogestrel, and 53 (69%) contained gestodene. The comparison of the main demographic and lifestyle characteristics between 77 OC users and 200 non–OC users are described in Table 1. Oral contraceptive users were not different from non–OC users with regard to age, BMI, university-level education, marital status, nulliparity, and coffee drinking. Oral contraceptive users were more frequently smokers (P=.005) compared with non–OC users. However, the average number of cigarettes per day (4.4) was low among smokers and did not differ according to OC use. Overall, only seven women were heavy smokers, defined as 10 or more cigarettes per day; two of them were OC users, and five were non–OC users (P=.69). Additionally, OC users and non–OC users did not differ in physical activity, meat, fish, vegetables, carbohydrate, and alcohol consumption (data not shown). Only 14% of the study women used multivitamin and mineral supplements, with no difference according to OC use.
Concentrations of blood biomarkers in OC users were compared with those in non–OC users as shown in Table 2. Oral contraceptive users had higher lymphocytes than controls. Median concentration of high-sensitivity CRP in OC users was almost fourfold higher than that of non–OC users (1.32 compared with 0.34 mg/L, P<.001). In contrast, concentrations of homocysteine and folates were not different. Concentrations of total cholesterol (P=.001) and HDL cholesterol (P<.001) were significantly higher in OC users than in non–OC users; however, the ratio of total to HDL cholesterol did not differ between OC and non–OC users. Interestingly, the ratio of LDL to HDL cholesterol was lower in OC users (P=.016). Concentrations of triglycerides (P<.001) were significantly higher in OC users. Bilirubin, albumin, and ALP were significantly lower in OC users. Other blood biomarkers were not different.
The percentage of subjects with high-sensitivity CRP concentrations less than 0.5, 0.5 to less than 1.0, 1.0 to less than 3.0, and 3.0 to less than 10.0 mg/L, was 15.6%, 20.8%, 32.5%, and 27.3% in OC users, and 61.5%, 18.0%, 11.0%, and 8.5% in non–OC users, respectively (Table 3). Very few women (n=5) had high-sensitivity CRP equal or more than 10 mg/L, three among the OC users and two among the non–OC users. No woman had high-sensitivity CRP more than 20 mg/L. The ORs for progressive cutoffs of high-sensitivity CRP are shown in Table 3. Interestingly, non–OC users were much more likely to show protective high-sensitivity CRP values compared with OC users (OR 8.65; 95% CI 4.39–17.1); however, they were similar to OC users for not having risky high-sensitivity CRP levels (0.5 to less than 1.0 mg/L). Remarkably, OC users compared with non–OC users had more frequently high-sensitivity CRP risk levels. Specifically, OC users had highly significant OR 3.9 for high-sensitivity CRP values 1.0 to less than 3.0 mg/L (cardiovascular disease intermediate risk), and OR 4.0 for high-sensitivity CRP values 3.0 to less than 10.0 mg/L (cardiovascular disease high risk). In addition, OC users were significantly more likely to have high-sensitivity CRP levels 3 mg/L or more (OR 4.1) and 5 mg/L or more (OR 2.9) than non–OC users. Finally, OC users were four times as likely to have high-sensitivity CRP levels 10 mg/L or more. However, this difference was not statistically significant, likely because very few women had very high high-sensitivity CRP concentrations in our healthy population. By contrast, OC users did not show higher frequency either of elevated homocysteine or low folate levels than non–OC users (Table 3), confirming findings on continuous values showed in Table 2. Further, Table 3 shows multivariable regression analysis for the variables 1) age, BMI, smoking, and WBC and 2) age, BMI, smoking, WBC, cholesterol, triglycerides, albumin, bilirubin, ALP, and lymphocytes; the adjusted ORs confirmed findings obtained with crude ORs. Also, adjustments for health behaviors did not affect ORs (data not shown).
Correlations of high-sensitivity CRP with other biomarkers in OC users and non–OC users are presented in Table 4. High-sensitivity CRP concentrations showed significant linear associations with BMI both in OC users (P=.006) and non–OC users (P=.004). High-sensitivity CRP showed a significant positive association with neutrophils and negative correlation with lymphocytes, HDL cholesterol, and albumin only in non–OC users. Conversely, high-sensitivity CRP was strongly positively associated with triglycerides in OC users (P=.005), but not in non–OC users. Inverse correlation of high-sensitivity CRP with albumin and bilirubin concentrations in OC users did not reach significance (Table 4). High-sensitivity CRP was not associated with homocysteine and folate either in OC users or in non–OC users. Finally, we found no correlation of high-sensitivity CRP with smoking and coffee consumption or with other health behaviors (data not shown).
Scientific evidence demonstrates an association between increased high-sensitivity CRP or homocysteine and risk of cardiovascular disease in women.4,5,17,18,23–25 These two biomarkers have been associated with endothelial damage, venous thrombosis, myocardial infarction, ischemic stroke, peripheral arterial disease, and sudden cardiac death.1,2,23 High-sensitivity CRP and homocysteine concentrations vary remarkably by race, ethnic group, age, and gender.5,26
So far, few data exist on high-sensitivity CRP and homocysteine blood levels in young, healthy, normal-weight women.27,28 Potentially, detailed knowledge of serum concentrations of these biomarkers in specific groups of women may allow the development of effective early primary preventive strategies for cardiovascular disease. To this aim, our study focused on healthy, normal-weight women of reproductive age. Our study found that third-generation pills have a major effect on high-sensitivity CRP levels such that OC users were four times more likely to have high-sensitivity CRP in the 1.0 to less than 3.0 mg/L range (at intermediate cardiovascular disease risk) and in the 3.0 to less than 10.0 mg/L range (at high cardiovascular disease risk) than nonusers. Interestingly, we also demonstrated that OC users were much less likely than nonusers to have protective high-sensitivity CRP levels less than 0.5 mg/L. Specifically, non–OC users were nearly 9 times more likely to have low protective levels of high-sensitivity CRP than OC users.
We observed that in young normal-weight women, high-sensitivity CRP was positively correlated with BMI, thus confirming observations performed in mostly overweight or obese women.29 Interestingly, lifestyle behaviors (including smoking, alcohol, and physical activity) were not related to high-sensitivity CRP in our study. As a consequence, our data suggest that the only modifiable risk factor to decrease high-sensitivity CRP in young, healthy, nonobese women may be limited to reduction of OC use. Increased average high-sensitivity CRP concentrations in OC users has also been observed by other authors.27,29–31 One of the largest European studies was performed by Döring and colleagues29 on 120 third-generation OC user women a decade older (average 33 years) and with higher BMI (average 24 kg/m2) than ours; in OC users mean high-sensitivity CRP concentrations were 3.4-fold higher than in non–OC users. Another study examined a population more similar in regard to age and BMI to ours comprising healthy young nonsmoking college women. This investigation showed that 31 hormonal contraceptive users had 2.7-fold higher average concentrations of high-sensitivity CRP than 27 nonusers. A limitation of this study was that several different types of OC pills were included.31 However, these authors did not assess the risk of cardiovascular disease by OC use using the relatively newly described ranges of high-sensitivity CRP. A recent Finnish study27 found a frequency of high-sensitivity CRP more than 3 mg/L of 35.3% in 295 OC users and 10.3% in 751 non–OC users, these values are close to those we found in our population. At variance with our study, frequency of protective levels of high-sensitivity CRP less than 0.5 mg/L were not examined, the control non–OC user group included women with levonorgestrel-releasing intrauterine device, and the type of OC was not specified.27
The pathways by which OC induces elevation of high-sensitivity CRP are elusive. Some authors observed that the increase of high-sensitivity CRP induced by hormonal treatments does not seem to follow the classical acute phase response profile.30,32 This finding is supported by our observation that only in non–OC users high-sensitivity CRP had a strong negative correlation with albumin and a positive correlation with neutrophils. Probably, estrogens have direct effects on hepatic CRP synthesis.30 It cannot, however, be refuted that estrogen-induced specific effects such as oxidative stress can contribute to increase CRP in OC users. In fact, estrogens may directly modulate hepatic synthesis of several factors at the transcriptional level and may also have various immunomodulatory effects, therefore predispose to thromboembolic events by stimulating inflammatory mechanisms.
In our study, third-generation OCs had no effect on homocysteine concentrations, in agreement with some,19,20,33 but not all studies,5,21 which examined modulation of homocysteine by hormonal treatments. This finding is of relevance in our opinion, because gynecologists much more frequently monitor homocysteine before and/or during OC use compared with high-sensitivity CRP, because of the potential vascular disease risk of elevated homocysteine. Venous thromboembolism is a serious adverse effect of OC use. From our findings it seems that the increased risk of venous thromboembolism in users of third-generation OCs is likely not due to homocysteine elevation.
We agree with previous observations that fasting serum triglycerides, and HDL cholesterol is increased by third-generation OC use.9 At variance with other studies,9,34 we did not observe decreased LDL cholesterol in OC users compared with controls. However, we observed a decreased ratio of LDL to HDL cholesterol in OC users compared with non–OC users. From our findings, third-generation OC use seems beneficial to the cholesterol profile in young women, although it causes triglycerides elevation, which is likely estrogen related.
Our study is limited by the nonrepresentative nature of our study sample, because the general female population has greater proportions of obese, low-social status, and older premenopausal women. In fact, low socioeconomic status has been shown to positively modify the association between overweight, abdominal obesity, cigarette smoking, and CRP.35 Thus, our observed elevation of high-sensitivity CRP levels with third-generation OC use may be even more pronounced among the general female population. Although we had adequate power to test the difference between high-sensitivity CRP levels by OC use even after adjustment for potential confounders, our study is limited by the sample size. Stratified analyses of women by levels of high-sensitivity CRP produced small groups and thus, although statistically significant, our odds ratios had wide confidence intervals. Therefore, larger studies are necessary to confirm our findings.
In conclusion, our study showed that many young, healthy, normal-weight OC users have elevated concentrations of high-sensitivity CRP and thus they are potentially at higher cardiovascular disease risk than nonusers. Because this low-grade inflammatory condition is asymptomatic, it is not normally recognized and/or investigated. This OC effect may have implications for development of cardiovascular disease and venous or other inflammatory diseases, including migraine.36 Oral contraceptive use did not affect homocysteine concentrations, thus the mechanism underpinning increased venous thrombovascular risk associated with third-generation OC pills is still in need of elucidation and may require the search for biomarkers other than homocysteine.
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