Discussion
non-alcoholic fatty liver disease
ACUTE PHASE RESPONSE, INFLAMMATION, LYSIS, NON-ALCOHOLIC FATTY LIVER DISEASE, INSULIN RESISTANCE, DYSLIPIDEMIA
This study showed that 9 weeks of EE-based COC use had a multifold effect on the serum proteome compared with EV-based COC or DNG-only preparation; the number of affected proteins after exposure to EE + DNG was 24-fold the number for EV + DNG and 120-fold that for DNG only. Few proteins changed during the use of EV + DNG and no changes were detected during the use of DNG only. The most-affected pathways were the complement system, acute phase response signaling, metabolism-related LXR&FXR/RXR activation and the coagulation system. The findings of the proteomic analysis were validated with fetuin-B ELISA and our previously reported CBG ELISA and SHBG immunoassay, showing that treatment with EE + DNG increased serum levels of fetuin-B, CBG and SHBG more than EV + DNG or DNG only.To our knowledge, this is the first study to explore the effects of hormonal contraceptives on serum proteins with an untargeted, discovery-type proteomic analysis. Previously, only one study has utilized a proteomic analysis method during contraceptive use (EE-containing COCs have been shown to increase low-grade inflammation, reflected by increased levels of C-reactive protein (CRP) (EE is a highly potent estrogen known to have a major effect on hepatic protein synthesis: EE increases SHBG, CBG and angiotensinogen multifold compared with estradiol (In the pathway analysis, as many as 19 proteins related to the complement system were altered during EE + DNG treatment, which predicts inhibition of this pathway. The complement system is a complex immunological system involving ∼30 interacting proteins that compose a cascade of activation steps leading to activation of inflammation, opsonization of pathogens and cells for destruction, and direct killing of target cells or microbes by lysis (Untargeted proteomic analysis also enables the identification of interesting novel targets for future research. Fetuin-B is a relatively understudied hepatokine associated with insulin resistance, dyslipidemia, and non-alcoholic fatty liver disease (The main strength of the present study is that this randomized trial is the first comparison of EE- and EV-containing contraceptives combined with the same progestin. Randomization was successful regarding the clinical characteristics but also at the proteome level, as reflected in the mixed clustering of baseline samples. Therefore, the changes observed during the trial are likely related to the intervention and not to chance. However, we acknowledge that the 9-week study period is relatively short for concluding long-term effects. Therefore, these findings should be further examined in longer trials with specifically targeted measurements. Proteomic analysis was not considered in the original sample size calculation, given that the analysis was not the primary outcome. On the other hand, as no previous data exist, estimating power for a discovery-type setup is extremely challenging. However, in the field of discovery-type proteomics, the groups of 14 are quite extensive, especially considering the trial with repeated measurements. Despite these limitations, our results give valuable insight into the extent of the differences between the estrogen types in COCs.To conclude, EE + DNG had a greater impact on the serum proteome than that of EV + DNG or the DNG-only preparation. The differences between EE and EV go far beyond the well-known, traditional estrogen actions. Therefore, EV could provide a preferable option to EE in COCs in the future. Moreover, although our findings need to be validated in further studies utilizing targeted methods, this study provides a valuable basis for future research and guides the development of hormonal contraceptives.