Currently, the scientific interest in the effects of sport on oxidative stress levels is increasing both for health and performance implications [1, 3]. Several authors emphasize that additional research will be required in this field [1, 3] because discrepancies exist among studies especially regarding antioxidant strategies. Antioxidant and pro-oxidant pathophysiological pathways of physical activity need to be elucidated, apparently, beneficial effects on oxidative stress and health are obtained when regular moderate training is practiced . On the contrary, acute exercise seems to increase oxidative stress, but intriguingly the oxidative stimulus is necessary to upregulate endogenous antioxidant defenses . Gender-specific studies are warranted to take into account sex differences in factors potentially modulating oxidative stress [1, 34, 35].
Our observational study was the first to explore continuous and categorized oxidative stress levels according to OC use in a sample of 144 ethnically homogeneous white young adult female athletes practicing various sport disciplines. We found that oxidative stress levels (evaluated by hydroperoxides) vary considerably according to OC use, the median value of FORT units was almost twofold higher in OC users than non-OC users athletes. Moreover, by evaluating stratified levels of hydroperoxides, we observed markedly elevated frequencies in OC users compared to non-OC users with crude OR = 42, and adjusted OR = 60 for the ≥310 FORT units threshold, and dramatically elevated crude OR = 252, and adjusted OR = 315 considering the ≥400 FORT units threshold. Our findings highlighted a remarkable increase of elevated oxidative stress in OC-user athletes that was apparently not associated with their lifestyles and alimentary habits; likely, in OC users, antioxidant foods’ effects could be overwhelmed by pro-oxidant effects of OCs. Conversely, in non-OC users, we found negative relationships of oxidative stress with some alimentary habits, specifically, chocolate and fish servings per week. Our findings are consistent with the known antioxidant properties of chocolate/cocoa  and fish (rich in n3-polyunsaturated fatty acids) . It is to mention that in our study, OC users had fewer chocolate servings per week than non-OC users, thus, the chocolate/cocoa effects will need further investigations.
In our study, we did not observe smoking and supplement use effects on oxidative stress levels in OC users and non-OC users. It is to be noted, however, that number of cigarettes smoked was very low (on average one cigarette per day) and that we asked participants to refrain from the use of supplements in the 24 h before blood testing; thus, it is very likely that some substances as vitamin C were washed-out. Particular effects of supplements will require specific further research.
In non-OC-user athletes only, our study highlighted a positive association between the FORT levels and BMI. Other authors found an association between BMI and systemic oxidative stress markers in the general population  and in active adults . Of note, in our study, none of the athletes were obese; thus, we demonstrated that even in normal weight subjects oxidative stress had a positive association with increased BMI, likely representing progressive elevation of accumulated fat. However, we did not observe a relationship between oxidative stress and BMI in OC users. It seems plausible that the induction of oxidative stress by OC use can overshadow the effects related to BMI. Further investigations are warranted to assess this issue.
In OC users, we found an inverse relationship of hydroperoxides (FORT units) with total defense capacity against free oxygen radicals (FORD units), that did not reach significant values in non-OC users. However, our data do not permit to infer causality, i.e., the present study cannot assess whether OC use directly increased ROS production that provoked formation of hydroperoxides and consumed antioxidant defenses, and/or whether OC use directly reduced antioxidant defenses, which became insufficient to neutralize free radicals, in turn provoking hydroperoxidation.
Our current findings are in line with the only previous study examining oxidative stress (by assessment of malondialdehyde and lipid peroxides) in female athletes taking OC . This study compared 12 female judoists using OC containing drospirenone and ethinylestradiol with 14 non-OC users, and found that OC users had significantly higher lipid peroxidation and lower antioxidant defense . However, given the small number of subjects, the study did not perform multivariate analysis. Another study in non-athletic women found increased lipid peroxides (+176 %, p < 0.001) and oxidized low-density lipoproteins (LDLs) (+145 %, p < 0.002) in 32 OC users compared with 30 non-OC users . A study in Belgian women aged 40–48 years found a significant increase of lipid peroxides in 209 OC users compared to 119 non-users of contraception . An interesting study investigating time-course of hydroperoxide elevation in women users of a low estrogen dose pill containing drospirenone demonstrated that oxidative stress increased significantly after only 1 week of OC use, remained constantly elevated during OC use, and returned to basal levels within 1 week of OC discontinuation . These results by Finco and colleagues  seem in line with our observation that hydroperoxides in OC users are not related to months of OC use.
Mechanisms leading to elevation of hydroperoxides by OC use still need to be clarified . It is plausible that catabolism of exogenous hormones by involving activities of P450 cytochromes (CYPs) provokes increased ROS production  and depletion of reduced glutathione [24, 25]. Future studies are warranted to assess if the observed blood rise of oxidative stress associated with OC use is estrogens and/or progestin related [22, 24, 41]. Interestingly, a recent study showed that in vitro estradiol treatment of cells resulted in a significant increase in lipid peroxidation . Contradictory findings were presented by other studies on pro- or antioxidant action of estrogens [22, 24, 43, 44]. Some evidence suggested that estrogens are inversely related to antioxidant defense, in particular, high estrogen levels were correlated to decreased blood superoxide dismutase (SOD) levels , but on the other hand, a study on female rats reported no relation of administered estrogen with SOD, but a positive relation with increased lipid peroxidation .
Further studies with increased number of female athletes will be necessary to evaluate the biochemical pathways of oxidative stress elevation in OC users.
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 . They have been introduced to reduce severe adverse effects of OC use, especially venous thromboembolism, and other cardiovascular diseases [17, 45]. However, these new OC preparations are still associated with the risk of myocardial infarction, thrombotic stroke, and venous thromboembolism [41, 46, 47]. The risk of venous thromboembolism associated with OC use is of particular concern and has been recently investigated in a total of 10,562 cases of thromboembolism by Vinogradova and colleagues ; exposure to OC containing desogestrel had increased risk OR of 4.28, cyproterone 4.27, drospirenone 4.12, gestodene 3.64, norethisterone 2.56, norgestimate 2.53, and levonorgestrel 2.38 in respect to no exposure to OCs in the previous year . In our study, we did not find statically significant differences in FORT and FORD units between users of OC containing desogestrel, cyproterone, and drospirenone versus other progestins; however, we observed a tendency to higher hydroperoxide values in the first group of OC users. Larger studies will be necessary to assess this relevant issue.
The combined oral contraceptive pill is appreciated by sportswomen not only for birth control efficacy but also because OC provides a consistent 28-day cycle eliminating cycle-length variability and menstrual irregularities [48, 49]. Data on OC use and exercise capacity are sparse . Some studies show that athletes on OCs experience a slight reduction in maximal aerobic capacity and endurance capability or perceive an increased fatigue [17, 19, 50].
It is generally believed that oxidative stress can affect negatively athletic performance and recovery [1, 3, 4]. By recent evidence, redox biomarkers measured in blood adequately reflect tissue redox status ; thus, the blood increased oxidative stress associated with OC use likely parallels increased free radicals also in muscle, implying potentially detrimental effects in sport performances .
Assessing the pro- and antioxidative stress effects of sport activity in the young female population may be complicated by several confounding factors , among these OC treatments can constitute a major confounder as shown by our present findings.
Elevation of oxidative stress implies several potential adverse effects including chronic diseases comprising cardiovascular disease (CVD), endothelial damage, thromboembolic events, and cancer [2, 52]. Notably, upper extremity thromboembolism in athletes , the so-called effort thrombosis, has been associated with use of hormonal contraception . Whether increased risk of thromboembolism in OC-user female athletes is mediated by the increased oxidative stress needs further investigations.
There are limitations in our study. We studied young adult white female athletes, and thus, we cannot generalize these results to older athletes or sportswomen with different ethnic backgrounds; sport activities of athletes were heterogeneous ranging from aerobic to mixed aerobic-anaerobic and to anaerobic activity, and from elite to non-elite competing level, however, we recently showed that OC effects on high-sensitivity C-reactive protein (hsCRP), a biomarker of inflammation, did not vary according to the sport discipline practiced by female athletes . We selected women taking monophasic combined (i.e., containing an estrogen plus a progestin) contraceptive pills, and we excluded other kinds of hormonal contraception; OCs were heterogeneous in type and amount of hormonal components although the majorities were OCs of third generation [18, 41]. We did not have detailed data about composition and dosing of potentially antioxidant supplements like vitamin E, C, and beta-carotene . The observational nature of our study makes it impossible to determine if OC plays a causal role in the pathogenesis of elevated oxidative stress; however, a study by other authors seems to indicate a causative role . In our study, the confidence intervals were somewhat large; however, highly significant results were obtained also after multivariate adjustments including several confounders. Finally, we evaluated oxidative stress by an assay measuring hydroperoxides (expression mainly of lipid peroxidation) [2, 24, 30], which constitutes only one of the possible indirect markers to assess oxidative stress status ; however, the FORT and FORD assays have been validated for clinical oxidative stress evaluation .
Strengths of the present study include assessment of oxidative stress and several lifestyles and alimentary habits, the homogeneous ethnic group, the rather narrow age range of competitive athletes, and strictly healthy subject inclusion.
Further studies have to be carried out to expand our observations including larger numbers of athlete of different sport disciplines and to better assess biochemical pathways related to oxidative stress elevation, the exact time-course of oxidative stress elevation according to OC use, the clinical significance, and the impact on athletic performance of this occurrence.