This is the first observational study assessing the relationships of oxidative stress (by hydroperoxides) and low-grade chronic inflammation (by hsCRP) levels by continuous and categorized serum values according to OC use in 290 Caucasian childbearing healthy women.
Oxidative stress
The median value of FORT Units was almost 2-fold higher in OC-users than in non-OC-users. We found a remarkable elevated frequency of high oxidative stress levels in healthy OC-users: by the cutoff value of ≥310 FORT Units 92.0% (crude OR=37, adjusted OR=100 compared to non-OC-users), and by the cutoff value of ≥400 FORT Units 77.0% (crude OR=209, adjusted OR=342). Evaluation of FORT Units relationships with other study parameters showed that oxidative stress apparently was not associated to OC-users lifestyles and alimentary habits. It appears that pro-oxidant effects of OCs likely overwhelmed antioxidant effects of lifestyle/alimentary good habits in OC-users. At variance, oxidative stress in non-OC-users was negatively associated with some alimentary habits, specifically chocolate, red meat and fish servings per week. Our findings are consistent with the known antioxidant properties of some foods (47-49). It is to mention that in our study fruits consumption had a negative correlation with oxidative stress in all the 290 women, however, in the distinct subgroups of 100 OC-users and 190 non-OC-users such negative correlation did not reach statistical significance, thus, fruits effects will require further research (48).
We did not found oxidative stress association with smoking and supplement consumption in the study women. The number of cigarettes smoked, however, was very low (on average <1 cigarette/day), moreover participants were asked to avoid supplements use in the day before blood testing. Interestingly, a study (30) performed in post-menopausal women showed that folate administration, in the form of 5-methyltetrahydrofolate (5-MTHF), can reduce blood oxidative stress as assessed by FORT values reduction. Particular effects of supplements and/or drugs will require specific further research.
Elevated BMI is known to increase systemic oxidative stress in the general population (50), and in active adults (32). Confirmatory with other studies, we found that BMI was positively correlated to the FORT levels in non-OC-user women only, but not in OC-users. A similar finding was seen previously in female athletes (17). Apparently, the strong OC induction of oxidative stress cancelled benefits deriving from reduced BMI.
Results of our current study performed on the general female population concur with a previous investigation evaluating oxidative stress in female athletes (17) founding that 42 OC-users had significantly higher hydroperoxides than 102 non-OC-users (OR=42, 95%CI=12-149, p<0.001). However, that study did not measured hsCRP (17).
A research conducted in 40-48 years old Belgian women found a significant increase of lipid peroxides in 209 OC-users compared to 119 non-users of contraception (22). A study comparing 32 OC-users with 30 non-OC-users found increased lipid peroxides (+176%, p<0.001) and oxidized LDLs (+145%, p<0.002) in the former group of women (20).
Interesting studies (21, 31) investigating the time-course of hydroperoxide elevation in women users of a low estrogen dose pill containing drospirenone demonstrated that oxidative stress increased significantly after only one week of OC use, remained constantly elevated during OC use, and returned to basal levels within one week of OC discontinuation, thus suggesting a causative role of OC use in increasing oxidative stress (31).
Mechanisms leading to elevation of hydroperoxides by OC are still not definitively characterized (31), however, some evidence point to oxidative hepatotoxicity of OC (12). P450 cytochromes (CYPs) catabolizing exogenous hormones can cause increased ROS production (51) and, in turn, hyper-production of free radicals could provoke depletion of antioxidant defenses such as depletion of reduced glutathione (31, 33). However, the role of estrogens and progestogens in OC induced oxidative stress is still debated (20, 31, 52). An in vitro study showed that beta-estradiol treatment of cells resulted cytotoxic through oxidative stress provoking significant increase in lipid peroxidation (53).
By recent evidence tissue redox status is adequately reflected by redox blood biomarkers (54), thus, the increased oxidative stress measured in blood associated to OC use likely parallels increased free radicals also in several body organs (55).
C-reactive protein
In the present study, OC-use significantly increased all risky levels of hsCRP, while provoking a loss of the protective levels below 0.5 mg/L. Specifically, OC-users were more likely to have hsCRP levels ≥1 mg/L (crude OR=7.49, adjusted OR=11.3); ≥2 mg/L (crude OR=6.64, adjusted OR=11.1); ≥3 mg/L (crude OR=6.71, adjusted OR=8.05) and ≥5 mg/L (crude OR=3.27, adjusted OR=3.07) than non-OC-users. Finally, OC-users were 9.95 times as likely to have hsCRP levels ≥10 mg/L. These results are consistent with previous studies performed in 77 third generation pill OC-users (23) and 53 OC-users athletes (24). Our results are consistent also with a large Danish study finding low-grade inflammation (hsCRP 3-10 mg/L) in 29.9% of OC-users compared to 7.9% in non-OC users (26).
The role of hsCRP attesting low-grade inflammation in women was highlighted by large studies: women who developed cardiovascular events had higher baseline hsCRP levels than control subjects, so that hsCRP was a strong independent risk factor for any vascular event (RR=4.8; 95%CI=2.3-10.1) and for myocardial infarction or stroke (RR=7.3; 95%CI=2.7-19.9) (56). Further studies confirmed the key role of chronic low-grade hsCRP in risk of future CVDs in women (39).
Recent evidence supports chronic inflammation (CRP values over 10 mg/L) as a mechanism of ovarian carcinogenesis (57). Interestingly, one study (57) showed that ever users of OC had an increased risk for ovarian cancer (OR=3.24, 95%CI=1.62-6.47). Regarding breast cancer (14), a recent review stated that safety of long-term OC use for BRCA1/2 mutation carriers is uncertain, thus, non-hormonal contraceptive methods should be discussed with those women (13). Notably, combined estrogen plus progestogen contraceptives are considered human carcinogens and classified in Group 1 by the International Agency for Research on Cancer (14) for the liver and bile duct, breast and uterine cervix cancer.
Combined oral contraceptives may affect the mediators of low-grade chronic inflammation with potential additive risk in women with polycystic ovary syndrome (PCOS); however clinical implications of OC use by PCOS patients need further studies (29).
Inflammation (58) and oxidative stress (34) have been implicated in the etiology of depression and disturbed sleep (59); in turn, OC use has been associated with depression (60). More longitudinal research is needed to improve the understanding of mechanisms induced by inflammation in OC-users possibly affecting the psyconeurological pathways (61).
Correlation between oxidative stress and hsCRP
Overall, our data highlighted a strong positive correlation of hsCRP with oxidative stress (p<0.001). Women with oxidative stress over 400 FORT units were eight time as likely to have hsCRP ³2.0 mg/L. So far, there has been a limited focus on this relationship specifically in women.
It is to highlight that causality cannot be inferred by our data, i.e., the design of our study does not allow to determine whether OC use directly increased ROS production (possibly via oxidative hepatotoxicity) that cause formation of hydroperoxides and then oxidative stress induces inflammation and/or whether OC use directly increase hsCRP that in turn provokes hydroperoxidation. It is to mention that the nuclear factors NF-kB/IkB are key molecular switches both in oxidative stress and inflammation pathways (36), thus, molecular mechanisms could activate at the same time oxidative stress and inflammation.
Many potential adverse effects are linked to elevation of oxidative stress and inflammation including thromboembolic events, endothelial damage, CVDs, and cancer (62, 63). The biochemical pathways of oxidative stress and inflammation elevation in users of combined contraceptive pills have, however, to be still completely elucidated.
Impact of the composition of the contraceptive pill
The progestin component of combined contraceptive pills may be important in determining side effects of OC use (2, 5, 19). Recent studies demonstrated that hormonal contraception can modify the redox status in the vasculature of women using combined contraceptive pills containing low doses of ethinyl-estradiol and progestin agents such as drospirenone (20, 64) or norethisterone (18).
New generations of OC pills are characterized by lower estrogen content and by newer progestins, like desogestrel, gestodene, cyproterone, and drospirenone with lower androgenicity than past generation pills (52). They have been introduced to reduce severe adverse effects of OC use, especially thromboembolism, and other cardiovascular diseases (11). However, these new OC preparations are still associated with the risk of pulmonary embolism, myocardial infarction, thrombotic stroke and VTE (2, 52). A meta-analysis of observational studies (6) found that all four generations of progestin were associated with an elevated risk of ischemic stroke, with a higher first-ever ischemic stroke risk associated with current OC use compared with non-current OC use (overall summary OR=2.47, 95%CI=2.04-2.99). The risk of ischemic stroke among current OC users decreased significantly with decreasing estrogen dose: OCs of ≥50μg ethinyl-estradiol (EE) had OR=3.28 (95%CI=2.49-4.32), 30-40 mg EE OR=1.75 (95%CI=1.61-1.89), 20 mg EE OR=1.56 (95%CI=1.36-1.79), whereas progestin only pills had not significant OR=0.99 (95%CI=0.71-1.37) (6).
A Danish study (2) examined 1,626,158 nonpregnant women, 15-49 years-old, with no history of CVD or cancer. As compared with non-users, current use of OCs was associated with an absolute increased risk of thrombotic stroke and myocardial infarction, ranging from 0.9 to 1.7 for OC containing 20 mg EE, and ranging from 1.3 to 2.3 for preparations with 30 to 40 mg EE, with relatively small differences in risk according to progestin type (2).
The risk of VTE associated to OC use is of particular concern and has been recently investigated in a total of 10562 cases of thromboembolism (5). In respect to no exposure to OCs in the previous year, exposure to OC containing desogestrel had increased risk with OR of 4.28, cyproterone 4.27, drospirenone 4.12, gestodene 3.64, norethisterone 2.56, norgestimate 2.53, and levonorgestrel 2.38 (5). Similarly, another study (65) found that the relative risk of VTE for combined oral contraceptives with 30‐35 μg ethinyl-estradiol and gestodene, desogestrel, cyproterone acetate, or drospirenone were similar and about 50‐80% higher than for combined oral contraceptives with levonorgestrel.
In our study we did not find statically significant differences in hsCRP values between users of second, third and fourth generation pills. However, second generation pill containing levonorgestrel had lower FORT values than third and fourth generation pills. Notably the group of OC containing desogestrel, cyproterone, and drospirenone had higher hydroperoxides values compared to all other progestins. Whether increased risk of thromboembolism in OC-users is mediated by the increased oxidative stress and/or chronic low-grade inflammation needs further investigations are necessary to assess this relevant issue.
Strengths and limitations
Limits of our study include: recruitment of young adult Caucasian females, and thus, results cannot be generalized to older women and/or to women with different ethnic backgrounds; only women taking monophasic combined contraceptive pills were included in the study, excluding other types of contraceptive drugs; OCs were heterogeneous in type and amount of hormonal components although the majorities were OCs of third generation; detailed data about composition and dosing of potentially antioxidant supplements like vitamin E, C, and beta-carotene were not registered (31, 33). Finally, oxidative stress was evaluated by an assay measuring hydroperoxides (expression mainly of lipid peroxidation) (31, 41, 63), that constitutes only one of the possible indirect markers to assess oxidative stress status (66); however, the FORT assays has been validated for clinical oxidative stress evaluation (30, 45).
Strengths of the present study include assessment of oxidative stress and hsCRP and several lifestyles and alimentary habits, the homogeneous ethnic group, the rather narrow age range of women, and strictly healthy subject inclusion.
Based on present data, increase of oxidative stress and hsCRP provoked by OC use poses the question of medical eligibility criteria for contraceptive use (67, 68), in particular for women affected by major diseases like high risk of CVDs, having had cancer or with major immune/inflammatory diseases like multiple sclerosis (69), diabetes (41, 70) or HIV infection (71, 72).