Estetrol Inhibits Endometriosis Development in an In Vivo Murine Model

Abstract

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus, and it is associated with alterations in the expression of hormone receptors and inflammation. Estetrol (E₄) is a weak estrogen that recently has been approved for contraception. We evaluated the effect of E₄ on the growth of endometriotic-like lesions and the expression of TNF-α, estrogen receptors (ERs), and progesterone receptors (PRs) in an in vivo murine model. Endometriosis was induced surgically in female C57BL/6 mice. E₄ was delivered via Alzet pump (3 mg/kg/day) from the 15th postoperative day for 4 weeks. E₄ significantly reduced the volume (p < 0.001) and weight (p < 0.05) of ectopic lesions. Histologically, E₄ did not affect cell proliferation (PCNA immunohistochemistry) but it did increase cell apoptosis (TUNEL assay) (p < 0.05). Furthermore, it modulated oxidative stress (SOD, CAT, and GPX activity, p < 0.05) and increased lipid peroxidation (TBARS/MDA, p < 0.01). Molecular analysis showed mRNA (RT-qPCR) and protein (ELISA) expression of TNF-α decreased (p < 0.05) and mRNA expression of Esr2 reduced (p < 0.05), in contrast with the increased expression of Esr1 (p < 0.01) and Pgr (p < 0.05). The present study demonstrates for the first time that E₄ limited the development and progression of endometriosis in vivo.

Keywords: apoptosis; endometriosis; estetrol; hormone receptors; mouse model; oxidative stress; proliferation.

Scheme 1

Experimental design. Surgical endometriotic-like lesion induction was made through an autologous transplant in female C57BL/6 mice. After 15 days of the establishment of endometriotic-like lesions, the treated group received subcutaneous mini osmotic pumps, releasing E₄ at a dose of 3 mg/kg/day solved in the vehicle (polyethylene glycol + dimethyl sulfoxide). Control group received vehicle-only. Over 4 weeks of treatment, animals were euthanized and samples were taken. EDT: endometriosis, and E₄: estetrol. The scheme was created in part using Servier Medical Art, provided by Servier, and licensed under a Creative Commons Attribution 4.0 unported license (https://creativecommons.org/licenses/by/4.0/).

Figure 1

Effect of E₄ on the growth of endometriotic-like lesions. In mice with induced endometriosis, the number of established lesions (a), their volume (b), and their weight (c) were examined after receiving either 3 mg/kg/day of E₄ (EDT+E₄) or vehicle-only (EDT+Veh). Mice were treated continuously for 4 weeks, from day 15 after endometriosis induction surgery to the day of euthanasia. Representative photography of endometriotic-like lesions treated with E₄ or vehicle after endometriosis induction (d). Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM (n = 8 animals per group). * = p < 0.05 and *** = p < 0.001. EDT: endometriosis; E₄: estetrol; and Veh: vehicle.

Figure 2

Effect of E₄ on cell proliferating in endometriotic-like lesions. (a) Micrography of representative slices from immunohistochemical analysis of PCNA in endometriotic-like lesions from endometriosis-induced mice treated continuously for 4 weeks with E₄ (EDT+E₄) or vehicle-only (EDT+Veh). The inset shows PCNA-negative control (NC) incubated without primary antibody. Magnification: 400×. (b) Percentage of PCNA-positive cells (brown nuclear reactivity). Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM from 4 to 6 representative visual fields (n = 6 animals per group). EDT: endometriosis; E₄: estetrol; and Veh: vehicle.

Figure 3

Effect of E₄ on cell death in endometriotic-like lesions. (a) Micrograph of representative slices from the TUNEL technique in endometriotic-like lesions from endometriosis-induced mice treated continuously for 4 weeks with E₄ (EDT+E₄) or vehicle-only (EDT+Veh). The inset shows TUNEL-negative control (NC) incubated without terminal deoxynucleotidyl transferase. Magnification: 400×. (b) Percentage of TUNEL-positive cells (brown nuclear reactivity). Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM from 4 to 6 representative visual fields (n = 6 animals per group). * = p < 0.05. EDT: endometriosis; E₄: estetrol; and Veh: vehicle.

Figure 4

Effect of E₄ on oxidative stress in endometriotic-like lesions. The activity of the antioxidant enzymes SOD (a), CAT (b), and GPX (c) and the levels of TBARS/MDA (d) were determined by spectrometry methods in endometriotic-like lesions from endometriosis-induced mice treated with E₄ (EDT+E₄) or vehicle-only (EDT+Veh) for 4 weeks. Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM (n = 8 per group). * = p < 0.05, and ** = p < 0.01. EDT: endometriosis; E₄: estetrol; Veh: vehicle.

Figure 5

Effect of E₄ on TNF-α expression. (a) Relative mRNA levels of Tnf were quantified by RT-qPCR in endometriotic-like lesions from endometriosis-induced mice treated for 4 weeks with E₄ (EDT+E₄) or vehicle-only (EDT+Veh). (b) TNF-α protein was quantified by ELISA in peritoneal fluid. Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM (n = 6 animals per group). * = p < 0.05. Tnf: tumor necrosis factor transcript variant 2; EDT: endometriosis; E₄: estetrol; and Veh: vehicle.

Figure 6

Effect of E₄ on expression of Esr2, Esr1, and Pgr. Relative mRNA levels of Esr2 (a), Esr1 (b), and Pgr (c) were quantified by RT-qPCR in endometriotic-like lesions from endometriosis-induced mice treated for 4 weeks with E₄ (EDT+E₄) or vehicle-only (EDT+Veh). Statistical significance was determined by Student’s t-test. All data are presented as mean ± SEM (n = 6–8 animals per group). * = p < 0.05, and ** = p < 0.01. EDT: endometriosis; E₄: estetrol; Veh: vehicle; Esr2: estrogen receptor beta gene; Esr1: estrogen receptor alpha gene; and Pgr: progesterone receptor gene.