A comprehensive study of the effects of medroxyprogesterone acetate, levonorgestrel and norethindrone on endometrial cells using proteomics and genomic analysis

S Mirkin, DF Archer

CONRAD, The Jones Institute, Norfolk, VA, United States

Introduction Progestin-only contraceptives provide a practical solution to family planning needs in developed and developing countries. Unfortunately, bleeding in the early months of treatment often results in the discontinuation of these otherwise safe and efficacious contraceptive methods. The cause and effect relationship between progestin-only contraceptives and irregular uterine bleeding remains unclear. Clinical and basic science studies have indicated that alterations in endometrial microvasculature may account for this disorder. We designed our study to investigate the effects of medroxyprogesterone acetate (MPA), levonorgestrel (LNG), norethindrone (NET) on angiogenic genes and proteins using microarray and proteomic technology, in Ishikawa cells.

Design Ishikawa Cells were treated with MPA, LNG, and NET. For genomic analysis, RNA was extracted. cDNA arrays were produced containing 2000 sequence of clones spotted. The tailor-made cDNA arrays included genes that are known to be important for endometrial angiogenesis. Labeled cRNA were hybridized to the array and the signal quantified. For proteomics analysis, SELDI was used. Protein lysates were loaded robotically on an IMAC chip. Each protein peak was labelled and its intensity was normalized for total ion current. Statistical analysis of the genomic data was performed using pair-wise multiple group comparison, and proteomic data was based on CART analysis.

Results Genomic analysis revealed that all progestins tested modified approximately the same number of genes (45%). MPA upregulated more genes related with angiogenesis and at higher extend (i.e. VEGF, EGF, ANG-1, TNF-a) than LNG and NET. Genes involved in menstrual bleeding (i.e. MMP2, MMP9, IL-1. MMP1) were also highly upregulated by MPA when compared with the others progestins. TSP-1 and TSP-2 genes with apoptotic activity on endothelial cells were also upregulated with MPA. SELDI spectra revealed a different proteomic fingerprint with each of the 3 compounds investigated. Two unique 7 and 10 kD protein peaks were observed in MPA treated cells.

Conclusion Genomic and proteomic analyses identified a differential gene and protein expression profile between MPA, NET and LNG in Ishikawa cells. MPA upregulated more angiogenic genes and at higher level than the other two progestins. SELDI revealed a differential effect at protein level of these 3 synthetics progestins confirming genomic findings. Based on these data we suggest that synthetic progestins may have a differential effect on endometrial angiogenesis and this may be related with difference rate in endometrial bleeding observed in the clinical setting.