title: Mass spectrometry identification of potential mediators of progestin-only contraceptive-induced abnormal uterine bleeding in human endometrial stromal cells.
abstract: OBJECTIVE
  Thrombin and hypoxia each target human endometrial stromal cells (HESCs) to mediate long-acting progestin-only contraceptive (LAPC)-induced abnormal uterine bleeding (AUB). Thus, the secretome resulting from treatment of primary cultures of HESCs with thrombin or hypoxia was screened by mass spectrometry (MS) to detect potential protein mediators that lead to AUB.
  
  
  METHODS
  Cultured HESCs were primed with estradiol±medroxyprogesterone acetate (MPA) or etonogestrel (ETO), the respective progestins in MPA-injected and ETO-implanted LAPCs, and then treated by incubation with thrombin or under hypoxia. Collected conditioned medium supernatants were used for protein identification and quantitation of potential AUB mediators by liquid chromatography combined with tandem mass spectrometry analysis. Microarray analysis of parallel cultures and immunostaining of endometrial biopsies of LAPC users vs. nonusers corroborated MS results.
  
  
  RESULTS
  MS identified several proteins displaying changes in expression levels from either thrombin or hypoxia treatments that are integral to angiogenesis or extracellular matrix formation. Several MS-identified proteins were confirmed by mRNA microarray analysis. Overexpressed stanniocalcin-1 (STC-1) was observed in endometrium of LAPC users. Unlike controls, all LAPC users displayed endometrial tubal metaplasia (ETM).
  
  
  CONCLUSIONS
  MS analysis identified many proteins that can affect angiogenesis or vessel integrity, thereby contributing to AUB. Confirmation of STC-1 overexpression in LAPC users and microarray data supports the validity of the MS data and suggests STC-1 involvement in AUB. The discovery of ETM in LAPC users indicates that LAPC-related side effects extend beyond AUB. The results presented here demonstrate a complex biological response to LAPC use.
  
  
  CONCLUSIONS
  MS identified several HESC secreted proteins deregulated by thrombin and hypoxia that may mediate LAPC-induced AUB. The revelation of overexpressed STC-1 by combined in vivo and in vitro observations identifies a potential target for future studies to prevent or minimize LAPC-induced AUB.
authors.affiliation: Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Shapiro
authors.affiliation: Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
authors.forename: Murat
authors.initials: M
authors.lastname: Basar
authors.affiliation: Department of Obstetrics and Gynecology, The University of South Florida, Tampa, FL 33606, USA.
authors.forename: Umit A
authors.initials: UA
authors.lastname: Kayisli
authors.affiliation: Department of Obstetrics and Gynecology, The University of South Florida, Tampa, FL 33606, USA.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Guzeloglu-Kayisli
authors.affiliation: Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
authors.forename: S
authors.initials: S
authors.lastname: Joseph Huang
authors.affiliation: Department of Pathology, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
authors.forename: Adrian A
authors.initials: AA
authors.lastname: Suarez
authors.affiliation: Biomedical Informatics Shared Resource, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
authors.forename: Hatice Gulcin
authors.initials: HG
authors.lastname: Ozer
authors.affiliation: Department of Obstetrics and Gynecology, The University of South Florida, Tampa, FL 33606, USA. Electronic address: fschatz@health.usf.edu.
authors.forename: Frederick
authors.initials: F
authors.lastname: Schatz
authors.affiliation: Department of Obstetrics and Gynecology, The University of South Florida, Tampa, FL 33606, USA.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Lockwood
mesh_terms: D002478:Cells, Cultured; D003271:Contraceptive Agents, Female / Q000008:administration & dosage / Q000009:adverse effects*; D017135:Desogestrel / Q000008:administration & dosage / Q000009:adverse effects; D004717:Endometrium / Q000166:cytology; D004958:Estradiol / Q000008:administration & dosage / Q000009:adverse effects; D005260:Female; D006023:Glycoproteins / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000139:chemically induced; D013058:Mass Spectrometry; D017258:Medroxyprogesterone Acetate / Q000008:administration & dosage / Q000009:adverse effects; D011372:Progestins / Q000008:administration & dosage / Q000009:adverse effects*; D017154:Stromal Cells / Q000187:drug effects*; D013917:Thrombin / Q000187:drug effects; D014592:Uterine Hemorrhage / Q000139:chemically induced*
chemical_list: D003271:Contraceptive Agents, Female; D006023:Glycoproteins; D011372:Progestins; C044815:etonogestrel; D004958:Estradiol; C051795:teleocalcin; D017135:Desogestrel; D017258:Medroxyprogesterone Acetate; D013917:Thrombin
keywords: Angiogenesis; Endometrial tubal metaplasia; Extracellular matrix; Mass spectrometry; Stanniocalcin-1