Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings

Abstract

BackgroundThe availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.MethodsFive independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.ResultsOf 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.ConclusionsCEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and fracture.Clinical Trials RegistrationClinicalTrials.gov identifier: NCT00000611

Confounding Effects of Hormone Replacement Therapy in Protein Biomarker Studies

“…However, there are numerous challenges associated with studying alterations in the serum/plasma proteome in relation to diseases such as cancer, in part due to exogenous factors that can alter protein levels even if issues related to sample collection, processing, and storage that can affect protein stability and levels are minimized. Recently we applied in-depth quantitative proteomics to determine the effects of post-menopausal hormone replacement therapy with estrogen and estrogen + progesterone on the serum proteome (1, 2). Using serum collections from the Women’s Health Initiative (WHI) randomized trials, large scale proteomic analyses were performed to compare proteins levels in serum collected from women at baseline and one year after HRT.…”

Menopause and Mitochondria

“…Other actions — including increased levels of inflammatory proteins and proteins that play roles in coagulation (Katayama et al, 2009) — may affect cognition adversely, leading to outcomes different from those anticipated on the basis of simpler in vitro experiments and in vivo models. These actions may account, for example, for increased incidence of ischemic stroke reported in some trials of hormone therapy (Rossouw et al, 2007).…”

Postmenopausal Hormone Therapy and the Risks of Coronary Heart Disease, Breast Cancer, and Stroke

“…Using an intact protein analysis system that reliably identifies and quantifies relative protein abundance for about 300-400 proteins, it was found 47,48 that there was nominally significant evidence of change between baseline and 1-year in blood concentrations of nearly half of the proteins quantified for women assigned to active hormone therapy. 48 This observation alone may suggest that a cautious approach is needed in the use of these potent regimens.…”