OCs modify the pituitary-ovarian axis (decreased luteinizing hormone, follicle-stimulating hormone, testosterone and estradiol), which is characteristic of the inhibition of ovulation [31, 32]. The pituitary-adrenal axis (increased cortisol) is also altered by OCs and this results is in line with previous findings [6, 33].
Thyroid-stimulating hormone levels were not significantly different between Fs and FOCs. However, when FOCA- and FOCA+ were considered, thyroid-stimulating hormone was elevated in FOCA-. Our results are in line with those observed by Wiegratz et al. , who reported that thyroid-stimulating hormone was significantly increased with the use of OCs containing a non-androgenic progestin .
We also confirmed that OCs induced variations in hematological and biochemical parameters, such as lymphocyte count, prothrombin time, total iron binding capacity of transferrin, C-reactive protein, and lipids [10, 23–25]. Some parameters were influenced by the androgenic properties of progestin: high-density lipoproteins were higher in FOCA-, in accord with the findings of van Rooijen et al. , while hemoglobin, white blood cell count, calcium, percentage saturation and γ-glutamyl transpeptidase were lower in FOCA-, as previously reported .
The primary novelty of our study is the fact that OCs modified estrogen receptor α and estrogen receptor β levels, and estrogen receptor β activity, while leaving androgen receptor expression unchanged. In particular, estrogen receptor α was markedly increased, whereas estrogen receptor β was largely decreased; consequently, the ratio of α/β was greatly altered. The variations in estrogen receptor levels were associated with changes in the activation status only of estrogen receptor β. In fact, estrogen receptor α activity was undetectable in all groups, indicating that this receptor is not active in basal conditions. Conversely, p38 phosphorylation, an important step in estrogen receptor β signal transduction , was significantly lower in FOCs than in Fs. Importantly, the androgenic and non-androgenic properties of progestin affected only the expression of the β isoform.
OC-induced modification of hormonal levels and the estrogen receptor α/estrogen receptor β ratio was accompanied by a significant increase in basal release of TNFα. When the ratio between the two estrogen receptors was the highest, we observed the greatest release of TNFα. Interestingly, these data strongly suggest that MDMs retain a selective 'memory' of their in vivo environment. They also suggest that FOCs who also had high C-reactive protein levels are more prone to inflammation. In this context, it is important to remember that FOCs had higher cortisol levels, which could impact release of TNFα. It should be noted that lipopolysaccharide-induced release of TNFα was higher in Fs and that Fs had lower cortisol and higher estradiol. The influence of sex hormones on release of TNFα has been suggested by Amory et al. , and recently a direct correlation between estrogen receptor α expression and the suppression of lipopolysaccharide-induced CXCL8 secretion has been shown , but because estrogen receptor α was not active in our samples, we believe that the cortisol increase is of some importance. These results are in line with the OC-induced modifications of human T lymphocytes . Notably, the release of cytokines by macrophages and monocytes appears to be an endocrine phenomenon. Generally, when estrogen is elevated, resting peripheral blood monocytes release less interleukin-1β and TNFα [38, 39]. Moreover, monocytes obtained from surgically postmenopausal women release a higher amount of cytokines, and importantly, the administration of estrogen restores premenopausal cytokine levels . When peripheral blood mononuclear cells are stimulated with lipopolysaccharide, mRNA expression and secretion of interleukin-1β and TNFα are increased in the luteal phase compared with the follicular phase .
Another important result of this study is the fact that OCs ameliorate endothelial function, as indicated by decreased asymmetric dimethylarginine. This marker of endothelial function is also an independent predictor of cardiovascular events and mortality . Although the absolute reduction of this endogenous inhibitor of nitric oxide synthase was small, the biological variation in the plasma asymmetric dimethylarginine/arginine ratio is also very low and even a slight increase in the asymmetric dimethylarginine/arginine ratio is associated with an elevated risk of acute coronary events . The reduction in the asymmetric dimethylarginine/arginine ratio is in line with the results of Valtonen et al. . Notably, the ratio increase was mainly sustained by a decrease in arginine. The decrease in arginine is not a universal finding , and the reasons for this discrepancy are not well understood, but a decrease in arginine has been reported after oral hormonal replacement therapy .
The reduction in total DNA methylation in FOCs was small but significant. Variations in DNA methylation imply heritable epigenetic changes in gene function . Notably, global hypomethylation predisposes to age-related chronic diseases, including atherosclerosis [43, 44]. The global hypomethylation of DNA and reduction of asymmetric dimethylarginine occurred in the presence of a significant variation of homocysteine; previously it has been shown that folate does not differ between OC users and non-users , suggesting that the decreases in asymmetric dimethylarginine and DNA methylation are not attributable to a decrease in folate. In our opinion, the low levels of cysteine and DNA methylation suggest a slowdown during the demethylation and trans-sulfuration phases of the methionine cycle, which can reasonably produce a decrease in asymmetric dimethylarginine, although it is not possible to exclude other mechanisms.
When the study population was stratified for OC use, the results obtained were mainly in line with results in the literature, indicating that the sample number was sufficient to discriminate differences. Indeed, the further stratification into FOCA+ and FOCA- groups might have influenced the statistical power, and therefore, further differences due to the activity of progestin may not have been detected. Some information was self-reported data, which contains several potential sources of bias. Another caveat is the lack of randomization and the fact that the study enrolled women who were treated with several OCs that contained different progestin-based molecules with androgenic and non-androgenic properties. However, the current study specifically focused on evaluating factors related to MDM function in a real population and whether function was affected by OC treatment, as well as investigating whether OCs could also produce alterations in cell functions, thereby affecting the pharmacodynamics of the drugs under examination.