All subjects were interviewed for their medical history, menstruation information, and smoking status. Measurements of body mass index, blood pressure and biochemical variables were performed in the clinic. Blood samples were obtained at the luteal phase confirmed by ultrasound, OCT users were taking 1.0 mg of NET at this time. Total cholesterol, triglyceride and high-density lipoprotein cholesterol were measured with standard enzymatic methods (Mitsubishi BCL Laboratory Co. Ltd., Tokyo, Japan) in blood samples obtained after an overnight fast. Hemoglobin A1c (HbA1c) was measured with high-performance liquid chromatography. The d-ROMs test for ROMs in blood was implemented by the Free Radical Analytical System (Diacron, Grosseto, Italy) according to the analytical manual.[7,8] The results of the d-ROMs test was expressed as Carr U (1 Carr U corresponds to 0.08 mg/dL of H₂O₂).[7] A previous report showed that replicate measurements on the same serum sample had an intra-assay coefficient of variation (CV) of less than 0.5% and an inter-assay CV of less than 2.9%.[9]

The mechanisms responsible for the results of the present study are unclear, but there are several possible explanations. The behavior of molecules related to oxidative stress in circulating blood and at the cellular level can differ in the types and dosages of estrogen and progestin within the treatments may be important. Estrogens show various cardiovascular actions on the endothelium, increasing the bioability of NO via genomic and non-genomic activation of NO synthase.[10] The most important difference between E2 and EE is that EE does not seem to protect endothelial function from oxidative stress.[11] A previous study found that the NO production in human ECV304-endothelial cells was increased by E2 in a dose-dependent manner but not by EE.[11] Similarly, the viability of endothelial cells after exposure to H₂O₂ was increased by E2 but not by EE, suggesting that EE does not protect endothelial cells from oxidative stress.[11] Another investigation reported that basal NO and prostaglandin production increased in cultured aortic cells from ovariectomized rats treated with E2, whereas NO and prostaglandin production were reduced in cells from non-ovariectomized rats treated with EE.[12] Moreover, a clinical study revealed that OCT significantly increased the plasma concentration of copper, selenium and lipid peroxides and decreased the levels of gamma-tocopherol and beta-carotene in women.[13] Taken together, these findings indicate that OCT can increase oxidative stress, possibly leading to vascular complications.

The administration of progesterone has anti-atherosclerotic effects with preferable lipoprotein profiles. Furthermore, progesterone may reduce ROS formation and cause vascular relaxation in a tissue-specific fashion;[14] however; progesterone antagonizes the vasoprotective effects of estrogen on anti-oxidant enzyme expression and function, and enhances NADPH oxidase activity and the production of ROS.[15] In OCT, a progestogen-only contraceptive implant was reported to have no negative effects on cardiovascular risk factors (e.g., C-reactive protein, total/high-density lipoprotein cholesterol ratio and NO), suggesting progesterone does not negatively impact cardiovascular risk factors in healthy young women.[16] The risk of venous thrombosis differs depending on the type of progestogen given in combination with EE, and the appropriate dose and type of progestin may reduce the adverse effects of OCT on cardiovascular risk factors.[17] Recently, the MEGA study indicated that the risk of venous thrombosis with OCT could be increased by different progestin up to 3 to 7 fold.[18] Thus, progestogen may also antagonize the beneficial effects of estrogen on vasodilation in OCT.