title: Oral contraceptives modify DNA methylation and monocyte-derived macrophage function.
abstract: BACKGROUND
  Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin.
  
  
  METHODS
  Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells.
  
  
  RESULTS
  As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs.
  
  
  CONCLUSIONS
  OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.
authors.affiliation: National Laboratory of Sex-Gender Medicine of the National Institute of Biostructures and Biosystems, Osilo, Italy. ilacampesi79@yahoo.it.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Campesi
authors.forename: Manuela
authors.initials: M
authors.lastname: Sanna
authors.forename: Angelo
authors.initials: A
authors.lastname: Zinellu
authors.forename: Ciriaco
authors.initials: C
authors.lastname: Carru
authors.forename: Laura
authors.initials: L
authors.lastname: Rubattu
authors.forename: Pamela
authors.initials: P
authors.lastname: Bulzomi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Seghieri
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Tonolo
authors.forename: Mario
authors.initials: M
authors.lastname: Palermo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Rosano
authors.forename: Maria
authors.initials: M
authors.lastname: Marino
authors.forename: Flavia
authors.initials: F
authors.lastname: Franconi