Uterine metabolism of progesterone (P4) after vaginal administration in an extracorporeal organ perfusion system.

Abstract

Objective: To outline the endometrial and myometrial metabolism of progesterone (P4) after vaginal application. The following metabolites of P4 were considered: 20α-hidroxy-4-pregnen-3-one (20α-DHP), 5α-pregnan-3,20 dione, and 5β-pregnan-3,20 dione (5α-DP), 3α,5α-allopregnanolone (A). Design: Using our extracorporeal uterine perfusion system, two sets of experiments were conducted with cold and -radioactive P4. P4 was applied on the rim of vaginal tissue remaining attached to the uterus. Samples of endometrial and myometrial tissue and venous outflow were obtained after 3 h, 6 h, 9 h, 12 h, 24 h and 48 h, using at least 3 uteri per time interval. P4 and P4 metabolites present in venous outflow and tissue samples were taken as reflectors of whole organ and regional metabolism of P4. Materials/Methods: 48 uteri obtained from women of reproductive age offered >3 completed experiments for each set of time interval experiments. Uteri were connected to the extracorporeal perfusion system as previously described and approximately 50 mCi of 3H-P4 was applied on the vaginal collar. Uterine perfusion was maintained as needed for each time interval. Samples of uterine tissue and venous effluent were washed, blotted, weighed and homogenized before an aliquot of the suspension was taken for protein measurement (Bradford, Bio Rad). The suspensions were then extracted and injected in HPLC (Hewlett-Packard 1090). Separation of P4 from its different metabolites was carried out on a Lichrosper-100 C18 125 × 4.6 mm (Merck, Darmstadt, Germany) while eluting with a tertiary mobile phase (CH3CN/THF/H2O 34/12/54) at room temperature and flow of 1 ml/min. Retention times for P4, 20α-DHP, 5α-DP and A were previously verified by nanogram injections of pure standards (Steraloids, USA) followed by UV detection. 15“ Fractions were collected and counted for in a β-counter after addition of 4 ml of scintillation cocktail. Radioactivity of the predetermined were considered for calculation. Further confirmation of P4 and P4-metabolites was carried out by gas-chromatography mass spectrometry (GC-MS) on HPLC fractions obtained from uteri perfuzed with cold P4 and treated as described above. Results: As described in table. % Endometrial (E) and Miometrial (M) steroids during perfusion at time intervals after P4 administration. Tabled 1Steroids3h6h9h12h24h48h% P (E)485,2183,3684,8291,5693,36% 20α (E)1,532,182,051,902,19% (E)−14,79−16,64−15,18−8,44−6,64% 5α (E)4,455,135,733,341,73% allo (E)8,819,337,403,202,72% P (M)89,086,679,981,983,384,1% 20α (M)3,43,53,13,53,03,2% (M)−11,0−13,4−20,1−18.1−16,7−15,9% 5α (M)2,73,77,16,65,66,1% allo (M)4,96,29,98,08,16,6 Open table in a new tab Conclusions: P4 is metabolized in the human uterus into three primary metabolites which may carry of all or part of P4 effects on the myometrium possibly through non-genomic properties of these metabolites.