Ethinylestradiol in combined hormonal contraceptive has a broader effect on serum proteome compared with estradiol valerate: a randomized controlled trial

Abstract

Study question: Does an estradiol-based combined oral contraceptive (COC) have a milder effect on the serum proteome than an ethinylestradiol (EE)-based COC or dienogest (DNG) only?

Summary answer: The changes in serum proteome were multifold after the use of a synthetic EE-based COC compared to natural estrogen COC or progestin-only preparation.

What is known already: EE-based COCs widely affect metabolism, inflammation, hepatic protein synthesis and blood coagulation. Studies comparing serum proteomes after the use of COCs containing EE and natural estrogens are lacking.

Study design, size, duration: This was a spin-off from a randomized, controlled, two-center clinical trial. Women (n = 59) were randomized to use either EE + DNG, estradiol valerate (EV) + DNG or DNG only continuously for 9 weeks.

Participants/materials, setting, methods: Participants were healthy, young, white volunteer women. Serum samples were collected before and after 9 weeks of hormonal exposure. Samples from 44 women were available for analysis (EE + DNG n = 14, EV + DNG n = 16 and DNG only n = 14). Serum proteins were analyzed by quantitative, discovery-type label-free proteomics.

Main results and the role of chance: Altogether, 446 proteins/protein families with two or more unique peptides were detected and quantified. The number of proteins/families that altered over the 9-week period within the study groups was 121 for EE + DNG and 5 for EV + DNG, while no changes were detected for DNG only. When alterations were compared between the groups, significant differences were detected for 63 proteins/protein families, of which 58 were between the EE + DNG and EV + DNG groups. The most affected functions during the use of EE + DNG were the complement system, acute phase response signaling, metabolism and the coagulation system. The results were validated by fetuin-B and cortisol-binding globulin ELISA and sex hormone-binding globulin immunoassay.

Large scale data: Data are available via ProteomeXchange with identifiers PXD033617 (low abundance fraction) and PXD033618 (high abundance fraction).

Limitations, reasons for caution: The power analysis of the trial was not based on the proteomic analysis of this spin-off study. In the future, targeted proteomic analysis with samples from another trial should be carried out in order to confirm the results.

Wider implications of the findings: The EE-based COC exerted a broader effect on the serum proteome than the EV-based COC or the DNG-only preparation. These results demonstrate that the effects of EE in COCs go far beyond the established endpoint markers of estrogen action, while the EV combination is closer to the progestin-only preparation. The study indicates that EV could provide a preferable option to EE in COCs in the future and signals a need for further studies comparing the clinical health outcomes of COCs containing EE and natural estrogens.

Study funding/competing interest(s): Funding for this researcher-initiated study was obtained from the Helsinki University Hospital research funds, the Hospital District of Helsinki and Uusimaa, the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Medical Association, the University of Oulu Graduate School, the Emil Aaltonen Foundation, the Swedish Cultural Foundation in Finland, the Novo Nordisk Foundation, Orion Research Foundation and the Northern Ostrobothnia Regional Fund. The funders had no role in study design, data collection and analysis, publishing decisions or manuscript preparation. T.P. has received honoraria for lectures, consultations and research grants from Exeltis, Gedeon Richter, MSD, Merck, Pfizer, Roche, Stragen and Mithra Pharmaceuticals. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose.

Trial registration number: ClinicalTrials.gov NCT02352090.

Trial registration date: 27 January 2015.

Date of first patient’s enrolment: 1 April 2015.

Keywords: acute phase signaling; coagulation; combined contraceptive; complement; estradiol valerate; ethinylestradiol; metabolism; proteome; randomized controlled trial.

Figure 1.

Flow chart and study preparations. Flow chart of the study (A) and hormone contents of the study preparations (B). The regimens were used continuously for 9 weeks. DNG, dienogest; EE, ethinylestradiol; EV, estradiol valerate.

Figure 2.

Hierarchical clustering analysis based on the protein compositions of serum samples during a 9-week use of combined oral contraceptive. Hierarchical clustering dendrogram of samples using the Euclidean distance measure (Y-axis) and the Ward clustering algorithm. The analysis shows that samples from the three study groups were mixed at baseline but after 9 weeks of use of the combined oral contraceptive preparations, the serum proteome profile clustered the participants according to preparation. The EE + DNG group in particular showed distinct clustering. The cluster was produced using MetaboAnalyst. DNG, dienogest; EE, ethinylestradiol; EV, estradiol valerate.

Figure 3.

Amounts of changing and unique proteins/protein families in serum from women taking a 9-week course of combined oral contraceptive. Venn diagram showing the numbers of changed proteins after within-group analysis. DNG, dienogest; EE, ethinylestradiol; EV, estradiol valerate. *Interleukin1 receptor accessory protein, fetuin-B, angiotensinogen and ICOS ligand and Ig-like domain-containing protein. **Ectonucleotide pyrophosphatase/phosphodiesterase family member 2.

Figure 4.

Pathway and network analyses of changing proteins during EE + DNG use. Pathway analysis of the 10 pathways most affected during 9-week use of ethinylestradiol + dienogest (EE + DNG). Graph shows category scores; ‘threshold’ indicates the minimum significance level (scored as −log(P-value) from Fisher’s exact test, set here to 1.25). A positive z-score (orange bar) predicts activation of the pathway, whereas a negative (blue bar) predicts inhibition. The analysis was performed with QIAGEN Ingenuity Pathway Analysis (IPA) software. The four pathways in bold text are depicted below as networks. FXR, farnesoid X receptor; LXR, liver X receptor; RXR, retinoid X receptor.

Figure 5.

Validation of proteomic analysis of serum from women taking a 9-week course of combined oral contraceptive. The fold changes in serum Fetuin-B, CBG and SHBG during the trial, as measured by ELISA and immunoassay. Box blots present the fold changes gained from the validation analyses, whereas the blue dotted lines show the fold changes from the proteomic analysis. The fetuin-B concentration increased especially during use of the combined oral contraceptive containing ethinylestradiol + dienogest (EE + DNG). One major fetuin-B outlier was removed from the EE + DNG group, which did not affect the statistical analysis. CBG and SHBG concentrations have been reported previously (Kangasniemi et al., 2022; Haverinen et al., 2022a). DNG, dienogest; EE, ethinylestradiol; EV, estradiol valerate; CBG, cortisol-binding globulin; SHBG, sex hormone-binding globulin. *P < 0.05; **P < 0.01; ***P < 0.001.