Oxidative Stress Is Increased in Combined Oral Contraceptives Users and Is Positively Associated with High-Sensitivity C-Reactive Protein

Information concerning the mechanisms underlying oxidative stress and low-grade inflammation in young healthy women predisposing eventually to future diseases is scarce. We investigated the relationship of oxidative stress and high-sensitivity C-reactive protein (hsCRP) in fertile-age women by oral combined contraceptive (OC) use. Caucasian Italian healthy non-obese women (n = 290; 100 OC-users; 190 non-OC-users; mean age 23.2 ± 4.7 years) were analyzed. Blood hydroperoxides, as oxidative stress biomarkers, were assessed by Free Oxygen Radical Test (FORT). Serum hsCRP was determined by an ultra-sensitive method (hsCRP). Markedly elevated oxidative stress (≥400 FORT Units) was found in 77.0% of OC-users and 1.6% of non-OC-users, odds ratio (OR) = 209, 95% CI = 60.9–715.4, p < 0.001. Elevated hsCRP levels ≥ 2.0 mg/L, considered risky for cardiovascular diseases (CVDs), were found in 41.0% of OC-users and 9.5% of non-OC-users, OR = 6.6, 95%CI 3.5–12.4, p < 0.001. Hydroperoxides were strongly positively correlated to hsCRP in all women (rs = 0.622, p < 0.001), in OC-users (rs = 0.442, p < 0.001), and in non-OC-users (rs = 0.426, p < 0.001). Women with hydroperoxides ≥ 400 FORT Units were eight times as likely to have hsCRP ≥ 2 mg/L. In non-OC-users only, hydroperoxides values were positively correlated with weight and body mass index, but negatively correlated with red meat, fish and chocolate consumption. Our research is the first finding a strong positive correlation of serum hydroperoxides with hsCRP, a marker of low-grade chronic inflammation, in young healthy women. Further research is needed to elucidate the potential role of these two biomarkers in OC-use associated side-effects, like thromboembolism and other CVDs.


Introduction
The biological pathways and the biomarkers associated with the side effects of hormonal contraception remain elusive and require additional research. Large studies and meta-analysis have assessed the association of oral combined contraceptive (OC) use with major complications, mainly cardiovascular diseases (CVDs) like arterial and venous thromboembolism (VTE), myocardial infarction, ischemic stroke, pulmonary embolism, peripheral arterial disease, and sudden cardiac death [1][2][3][4][5][6][7][8][9][10][11] and also some types of cancer including breast, uterine cervix, as well as liver and bile duct cancers [12][13][14]. Moreover, a higher risk for preterm birth in women who had used OCs before pregnancy compared

Design and Setting of the Study
The study was conducted on healthy Italian adult women enrolled in the years up to September 2019; none of the volunteers were athletes, to avoid confounding effects of physical exercise [25].
All participants attended once the Medical Department laboratory (University of Udine, Italy) early in the morning (8-10 am) after 12 h fasting. A finger capillary and a venous blood withdrawal [24,25] was thus taken on seated subjects [18,45]. The day before, volunteers were asked to refrain from alcohol and supplement consumption as well as from physical activity. Menstrual cycle phase was not accounted for; however, menstruation bleeding days were avoided (days 1-7 of menstrual cycle) because during those days oral contraception consists of drug discontinuation or placebo use. Clinical, demographic, and habit data were blinded to personnel executing the blood collection and samples measurements.

Population
Women (age range 18-46 years) were recruited consecutively; announcements at Udine University were used to enroll the volunteers. All study participants filled questionnaires and volunteered for a venous and a finger capillary blood withdrawal. The inclusion criteria were: (a) no acute (current/recent) infection or chronic disease such as diabetes mellitus, autoimmune diseases, thyroid or cardiovascular disease, or any tumor [24,25]; (b) absence of pain from whatever cause [46]; (c) no assumption of anti-inflammatory drugs or antibiotics in the last 2 weeks [24,25], (d) no hormonal drugs other than monophasic combined contraceptive pills [24,25], (e) not lactating, pregnant, or postmenopausal [47]; (f) no present or past menstrual dysfunction [48]; and (h) no major sleeping disorders [18]. Eligible women for inclusion in the OC-users group were those using a monophasic combined oral contraceptive for at least 3 months, whereas for inclusion in the non-OCusers control group, women had never used hormonal contraception or had discontinued since at least 3 months any hormonal treatment [18,25]. Three-hundred women were examined for eligibility; at the time of blood withdrawal, 10 women were excluded due to acute disease potentially affecting the inflammatory status. A total of 290 participants were thus included in the study (n = 100 OC-users and n = 190 non-OC-users).
Demographic factors, medical history and lifestyle habits (including smoking) were collected by a questionnaire. Moreover, a 2 weeks long diary allowed to collect alimentary habits in the form of daily number of servings as already described [18]. Alimentary supplements consisted of a wide variety of commercial products containing variable amounts of single or mixed compounds, assumed by women occasionally, intermittently, or continually. Thus, alimentary supplements were not categorized into specific subgroups, and a categorical variable (yes or no) designed "supplement use" was employed for statistical analysis. Body mass index (BMI) was calculated as body mass (kg) divided by the square of stature (m).

Ethical Considerations
Written informed consent was obtained from each participant before enrolment and no compensation or incentive was paid to the participants for this study. The study was approved by the Institutional Ethical Committee of the Department of Medicine (University of Udine, number of ethical approval 554), and was conducted according to the rules of the Declaration of Helsinki (1975).

Oxidative Stress Assessment
Blood hydroperoxides, mainly consisting of lipid hydroperoxides [18,45], were determined in 20 µL of capillary blood using the Free Oxygen Radical Test (FORT assay; Callegari, Parma, Italy), a 6 min long colorimetric assay based on the ability of transition metals to catalyze the breakdown of hydroperoxides (ROOH) into radicals, according to the Fenton reaction [45]. Results were expressed as FORT Units, whereby 1 FORT Unit corresponded to 0.26 mg/L H 2 O 2 [45]. Variations of the intra-and inter-assay were both <5.0%. The range limits of measurable values were ≤160 and ≥600 FORT Units. Four non-OC-users had FORT values ≤ 160 and were set at 160 FORT Units, whereby 9 OC-users had ≥600 FORT Units and were set at 600 FORT Units for statistical calculations.
The threshold of 310 FORT Units was used as high cut-off level of hydroperoxides [18], and a value of 400 FORT Units was employed as very high threshold level of oxidative stress [49].

C-Reactive Protein Measurement
Just after collection, venous blood was processed for serum separation as already described [25].
Serum hsCRP was measured on Olympus AU5400 biochemistry analyzer (Olympus Diagnostic Systems Group, Brea, CA, USA) using an ultra-sensitive immunoturbidimetric assay (Olympus System CRP latex). Detection limit of the assay was ≤0.1 mg/L. Samples with values below the detection limit were set at 0.1 mg/L for statistical calculations. The intra-and inter-assay variations were 1.1%, and 4.0%, respectively. Stratification of hsCRP levels were performed at 0.5 mg/L as no risky level, and 2.0 mg/L and 3 mg/L as risky levels for future CVD, as described [24,39,41].

Statistical Analysis
Normality of distribution of variables was assessed by the Kolmogorov-Smirnov test. Gaussian distributed data were shown as mean ± standard deviation (SD). The FORT and hsCRP data were not normally distributed, thus, their values were shown as median and interquartile range (IQR, 25th to 75th percentile) and non-parametric tests were used. Continuous variables were compared by the Mann-Whitney U-test. The comparison of proportions between groups was assessed by Pearson's X 2 -test that was appropriate for all the categorical variables except for "nulliparity" for which Fisher's exact test was used. Crude odds ratios (ORs) and 95% confidence intervals (CIs) were evaluated for categorical variables. Logistic regression was performed to evaluate the difference in oxidative stress between groups after adjustment for: age, BMI, smoking, supplement use, chocolate, fresh vegetable, fruits, red meat and fish servings per week. Bivariate relationships were evaluated by Spearman Rho test (r s ). All tests were 2-sided. Statistical significance was set at p values < 0.05. At an alpha level of 0.01, we had over 95% power to detect a difference in the proportion cases with hsCRP ≥ 2 mg/L between the OC-users and the non-OC-users groups and to find a correlation between hsCRP and FORT Units with r s > 0.4. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows, SPSS Inc., Chicago, IL, USA).

Results
On average, the 290 study women were 23.2 ± 4.71 year-olds, had BMI 21.2 ± 2.31 kg/m 2 , 97.9% were nulliparous, mostly enrolled at or employed by Udine University, 82.8% had university level education, 16.6% were smokers, and all had a middle-class socioeconomic status. Main demographic and lifestyle characteristics and continuous values of oxidative stress and hsCRP of the 100 OC-users and the 190 non-OC-users are summarized in Table 1.
A normal weight range (BMI 18.0-25.0 kg/m 2 ) was found in 91.4% of women, 9 women were underweight (3.1%, BMI < 18.0 kg/m 2 ), and 16 were overweight (5.5%, BMI > 25.0 kg/m 2 ); none of the women, however, had a BMI ≥ 30.0 kg/m 2 . OC-users had slightly more frequently a university education (89.0%) than non-OC-users (79.5%), p = 0.041 (Table 1). However, most of the studied parameters were not significantly different between OC-users and non-OC-users including smoking habits, coffee and nutritional supplement use. In our sample, women smoke on average less than 1 cigarette per day. Only one woman smoked 26 cigarettes per day, none of the other smokers smoked more than 15 cigarettes per day. Table 1 shows median (25th to 75th percentile) values of hydroperoxides that were 1.7fold higher in OC-users compared to non-users (median 472 vs. 270 FORT Units, p < 0.001). Values of hsCRP were 3.7-fold higher in OC-users compared to non-users (median 1.31 vs. 0.35 mg/L, p < 0.001).

Combined Oral Contraception
A hundred women were using OC from 3 to 144 months. Continuous values of FORT Units and hsCRP concentration were not correlated with months of OC use (p > 0.05 for both).

Stratified Values of Oxidative Stress and hsCRP
Regarding stratified hsCRP levels, the percentage of subjects with risky hsCRP concentrations was significantly higher in OC-users compared to non-OC-users; for the cutoff value of ≥2 mg/L crude OR 6.64, adjusted OR 11.1, and for the cutoff value of ≥3 mg/L crude OR 6.71, adjusted OR 8.05. Conversely, non-OC-users were much more likely to show protective hsCRP < 0.5 mg/L values compared to OC-users (OR 8.60; p < 0.001). Very few women (n = 6) had hsCRP ≥ 10 mg/L, 5 among the OC-users, and 1 among the non-OC-users. No woman had hsCRP ≥ 20 mg/L.
Women having elevated hsCRP values were much more likely to show elevated oxidative stress (Table 4). Specifically, among a total of 59 women having risky hsCRP ≥ 2 mg/L, 49 (83%) had FORT ≥ 310 Units and 38 (64%) had FORT ≥ 400 Units; furthermore, among a total of 46 women having hsCRP ≥ 3 mg/L, 39 (85%) had FORT ≥ 310 Units and 32 (70%) had FORT ≥ 400 Units. On the contrary, women with low protective levels of hsCRP (<0.5 mg/L) were much less likely to have elevated oxidative stress. Indeed, among the 134 women having protective hsCRP < 0.5 mg/L, only 26 (19%) had FORT ≥ 310 Units and 7 (5.0%) had FORT ≥ 400 Units. Two thresholds of elevated hydroperoxides levels (≥310 or ≥400 FORT Units) were compared with the corresponding low hydroperoxides levels (<310 or <400 FORT Units, respectively) in the whole group of 290 women. OR, odds ratio; CI, confidence interval; p value by two-tailed chi-squared test. Statistically significant differences are highlighted by bold characters.
By considering the two elevated thresholds of oxidative stress, namely FORT ≥ 310 or ≥400 Units (Table 4), women were roughly 8-fold more likely to have elevated hsCRP levels.

Discussion
The main outcomes of the present study are: (1) Elevated oxidative stress levels were found in non-athlete OC-users belonging to the general population of healthy young women; (2) OC-use resulted in chronic inflammation, confirming previous studies performed in lower number of women or different ethnic groups; (3) Oxidative stress and hsCRP were strongly correlated; (4) Body mass (as well as BMI) and some alimentary habits modulated oxidative stress in non-OC-users only.

Oxidative Stress
Oxidative stress was evaluated by an assay measuring hydroperoxides (expression mainly of lipid peroxidation) [33,45,50], that constitutes only one of the possible indirect markers to assess oxidative stress status [51]; however, the FORT assay has been validated to assess oxidative stress in human blood by various recent studies [18,32,45,52]. Specifically, the FORT assay has been validated for clinical use in women and has the advantage to be easily used in outpatient facilities [32,52]. Lewis, et al. [52] determined a value for individual biological variations of the FORT assay equal to 5.0%.
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). The median of continuous FORT Units was almost 2-fold higher in OC-users than in non-OC-users. In our study, among OC-users oxidative stress was not associated to their lifestyles and alimentary habits according to the study parameters. 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 [53][54][55]. 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 [54].
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 [32] 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 [56].
Elevated BMI is known to increase systemic oxidative stress in the general population [57], and in active adults [34]. Confirmatory with other studies, we found that BMI was positively correlated to the FORT levels in non-OC-user women, but not in OC-users. A similar finding was seen previously in female athletes [18]. 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 [18] 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 [18].
Consistently with the present study, a research conducted in 40-48 years old Belgian women found a significant increase of lipid peroxides in 209 OC-users compared to 119 nonusers of contraception [23]. Additionally, a study comparing 32 OC-users with 30 non-OC-users found increased lipid peroxides (+176%, p < 0.001) and oxidized LDLs (+45%, p < 0.002) in the former group of women [21].
Interesting studies [22,33], 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 [33].
Mechanisms leading to elevation of hydroperoxides by OC are still not definitively characterized [33], however, some evidence point to oxidative hepatotoxicity of OC [12]. P450 cytochromes (CYPs) catabolizing exogenous hormones can cause increased ROS production [58] and, in turn, hyper-production of free radicals could provoke depletion of antioxidant defenses such as depletion of reduced glutathione [33,35]. However, the role of estrogens and progestogens in OC induced oxidative stress is still debated [21,33,59]. An in vitro study showed that beta-estradiol treatment of cells was cytotoxic through oxidative stress inducing a significant increase in lipid peroxidation [60].
By recent evidence tissue redox status is adequately reflected by redox blood biomarkers [61], thus, the increased oxidative stress measured in blood associated to OC use likely parallels increased free radicals also in several body organs [62].
The role of hsCRP attesting low-grade inflammation in women was highlighted by large studies (41,58). An American study demonstrated that 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) [63]. Further studies confirmed the key role of chronic low-grade hsCRP for risk of future CVDs in women [41].
Recent evidence supports a role of chronic inflammation for female cancers [14,64,65]. 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.
Moreover, 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 [30].
New evidence suggests that inflammation [16] and oxidative stress [36] are implicated in the aetiology of depression and disturbed sleep [66]; in turn, OC use has been associated with depression [17]. More longitudinal research is needed to improve the understanding of mechanisms induced by inflammation in OC-users possibly affecting the psyconeurological pathways [67].

Correlation between Oxidative Stress and hsCRP
Overall, our data highlighted a strong positive correlation of oxidative stress with hsCRP (p < 0.001) in healthy young women; women with oxidative stress over 400 FORT Units were about eight time as likely to have hsCRP ≥ 2.0 mg/L. So far, there has been a limited focus on this relationship specifically in women without pathologies.
There is scientific evidence demonstrating elevation of oxidative stress and inflammation in several pathological conditions, particularly of vascular nature [68]. Correlation between hsCRP and blood markers of oxidative stress was observed for instance in acute myocardial infarction [69] and in patients with high risk of CVD [70]. The novelty of our study is finding such a correlation in healthy subjects, suggesting that some general physiological mechanism links oxidative stress and low-grade inflammation even in the absence of evident pathologies. Such effects are likely mediated by factors like nuclear factor kappalight-chain-enhancer of activated B cells (NF-kB) and nuclear erytroid 2 like factor-2 (Nrf2), two main transcription factors modulating several genes implicated both in oxidative stress and inflammation, through a complex balance among a variety of factors [71].
In our study causality cannot be inferred, because the design of the study does not allow to determine whether oxidative stress induces inflammation and/or whether the increase of hsCRP provokes hydroperoxidation. It is to mention, however, that the nuclear factors NF-kB and Nrf2 are key molecular switches both in oxidative stress and inflammation pathways [38,71], thus, molecular mechanisms could activate at the same time oxidative stress and inflammation.
In OC-users we found a higher frequency of elevated oxidative stress and inflammation compared to non-OC-users. dos Santos and colleagues [31] hypothesized that several mechanisms, including oxidative stress, elevate inflammation in hormonal contraception users, suggesting a causative role of oxidative stress in blood hsCRP increase. The biochemical pathways linking oxidative stress and inflammation elevation have, however, to be still completely elucidated, in particular in view of their potential adverse effects, including for example thromboembolic events, endothelial damage, CVDs, and cancer [50,72].

Impact of the Composition of the Contraceptive Pill
Accumulating evidence suggests that the progestin component of combined contraceptive pills may be important in determining the side effects of OC use [2,5,20]. Specifically, 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 [21,73] or norethisterone [19].
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 [59]. They have been introduced to reduce se-vere 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 [2,59]. A metaanalysis of observational studies [6] found that all four generations of progestin were associated with an elevated risk of ischemic stroke, and 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 ≥50 µg ethinyl-estradiol (EE) had OR = 3.28 (95%CI = 2.49-4.32), 30-40 µg EE OR = 1.75 (95%CI = 1.61-1.89), 20 µg 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 µg EE, and ranging from 1.3 to 2.3 for preparations with 30 to 40 µg 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 10,562 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 [74] 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. A relevant issue for future investigations is to assess whether increased risk of thromboembolism in OC-users is mediated by the increased oxidative stress and/or chronic low-grade inflammation.

Strengths and Limitations
Limitations of our study include the 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 β-carotene were not registered [33,35].
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. Of note, all study women were recruited before September 2019, thus avoiding any possible confounding by unrecognized SARS-Cov2 infection, although in our opinion it will be interesting in the future to explore the role of hormonal contraception in SARS-Cov2 infection.

Conclusions
This study adds to the existing evidence that OC use alters the oxidative homeostasis and modifies low-grade inflammatory status of young women. We found very high oxidative stress in the vast majority of OC-users (77% of them had FORT ≥ 400 Units). The strong positive relationship, that we observed in healthy women, between oxidative stress and basal chronic inflammation has several potential implications. Elevation of both parameters can potentially concur in OC side effects like the increased risk of CVDs, cancer and other diseases, posing the question of medical eligibility criteria for contraceptive use [75,76], 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 [77], diabetes [45,78] or HIV infection [79,80]. It remains to be determined whether oxidative stress or hsCRP elevation is the main driver of CVD risk and whether these two conditions are synergistic.
We also investigated lifestyle and alimentary habits of study women. OC-users appear resilient to antioxidant food and low BMI protective effects. However, in our opinion, future studies on food supplements having antioxidant and anti-inflammatory roles [54,56,81,82] could be designed and especially targeted for OC-user women. Based on our data, concurrent use of pro-oxidant [82] and/or proinflammatory [83] drugs in addition to OC use should also be carefully taken into account, and will require future investigations. We think advisable that, at least in women with pathologic conditions and/or at high risk of developing a pathology, oxidative stress and hsCRP should be monitored over OC time use.
Author Contributions: S.C. and M.P.F. designed the study, equally contributed to the study by performing most of experiments, analyzed the data, and wrote the manuscript. S.X. and C.B. helped in the design of the study and enrolled the study subjects; C.C., M.B. and G.B. contributed to the study by performing experiments. S.C. supervised the project. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Written informed consent was obtained from each participant before enrolment and no compensation or incentive was paid to the participants for this study.

Data Availability Statement:
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments:
We thank Patrizia Nacci, Luca Bazzichetto, and Silvia Lolini for technical support and Luigi Xodo for critical revision of the manuscript, Department of Medicine (University of Udine).

Conflicts of Interest:
The authors declare that they have no competing interests.