OBJECTIVE:The major cause of prematurity is preterm birth (PTB), associated with intrauterine inflammation. Defects in the Notch pathway harm placentation, and there is evidence between Notch activation and the inflammatory environment. In the action of PTB, surfactant A (SP-A) may have a pro-inflammatory or anti-inflammatory effect, and increased synthesis of prostaglandins illustrates their crucial roles in gestational tissues at parturition. Altogether, the potential of SP-A and prostaglandin inhibitors to prevent PTB through the placenta is worth exploring. This study evaluates the preventive effect of SP-A and Indomethacin (IND) treatment on placental inflammation in the LPS-induced PTB model.MATERIALS AND METHODS: Forty-eight female CD-1 mice were distributed to pregnant control (PC), Sham, PBS, IND (2 mg/kg; intraperitoneally), LPS (25mg/100ml; intrauterine), LPS+IND, SP-A block (SP-A B; 20ug/100ml; intrauterine) groups. The injections were performed on day 14.5 of pregnancy. Placentae were removed on day 15.5 of pregnancy, and immunohistochemical analyzes were performed. Differences in staining intensities between the groups for Cox-1, Notch-1 (N1), Dll-1, Jagged-2 (Jag-2), SP-A, Tlr-2, and Tlr-4 proteins were compared using ANOVA and Sidak's Multiple Comparison test. P values <0.05 were considered statistically significant. RESULTS: PTB rates were; 100%, 66% (in this group, delivery delayed for about 5 hours), and 50% in LPS, LPS+IND, SP-A B groups, respectively. LPS application caused damage to fetal and maternal vascular structures in the placenta, especially in the labyrinth zone (LZ). Placental volume decreased, and lymphocyte infiltration was observed. The morphological distinction between the compartments was unclear. N1 expression increased in both the junctional zone (JZ) and LZ. Cox-1 expression in the LZ decreased significantly (p<0.05), while the expression of N1, Dll-1, and Jag-2 increased significantly (p<0.05). Tlr-2 and Tlr-4 expression increased significantly in LZ and JZ, respectively. In the LPS+IND group, the LZ morphology was similar to the control, and placenta zone boundaries were distinguishable. In the LPS+IND group, N1, Jag-2, and Tlr-4 expression decreased significantly (p<0.05). In the SP-A B group, Cox-1 expression increased significantly (p<0.05).CONCLUSIONS: In the PTB model, Notch signaling, SP-A, and prostaglandin-associated signaling are disturbed in the maternal-fetal exchange site, the LZ and hormonal production site, the JZ of the placentae. While SP-A modulates the LPS-induced inflammatory response related to PTB, IND can prevent PTB via decreasing inflammation in the LZ.IMPACT STATEMENT: Activation of inflammatory signaling pathways can cause damage to the placenta during inflammation-related PTB. Our results highlight the necessity of future clinical studies utilizing prostaglandin inhibitors to improve the placental function in preventing this process.

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Objectives: Formononetin is one of the phytoestrogens that functions like a selective estrogen receptor modulator (SERM). In this study, we evaluated the effects of formononetin on endometriosis progression in vitro and in vivo. Materials and methods: After pathological confirmation, 10 eutopic and ectopic endometria were collected from patients with endometriosis. Ten eutopic endometria samples were collected from patients who did not have endometriosis. To determine the cytotoxic dose and therapeutic dose of formononetin, the concentration of 70% of the cells that survived after formononetin administration was estimated using a Cell counting kit-8 (CCK 8) assay. Western blot analysis was used to determine the relative expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 in the eutopic endometria without endometriosis, eutopic endometria with endometriosis, and ectopic endometria with endometriosis as the formononetin concentration was increased. We confirmed the effect of formononetin on apoptosis and migration in endometriosis using fluorescence-activated cell sorting (FACS) and wound healing assays, respectively. A mouse model of endometriosis was prepared using a non-surgical method, as previously described. The mice were intraperitoneally administered formononetin for four weeks after dividing them into control, low-dose formononetin (40 mg/kg/day) treatment, and high-dose (80 mg/kg/day) formononetin treatment groups. All the mice were euthanized after formononetin treatment. Endometriotic lesions were retrieved and confirmed using hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining of p27 was performed. Results: We set the maximum concentration of formononetin administration to 80 μM through the CCK8 assay. Based on formononetin concentration, the expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 proteins were measured using Western blot analysis (N = 4 per group). The expression level of pERK, p27, and pSTAT3 in eutopic endometrium with endometriosis tended to decrease with increasing formononetin concentration, and a significant decrease was noted at 80 μM. The expression of p27 in ectopic endometrium with endometriosis was also significantly decreased at 80 μM of formononetin. FACS analysis revealed that formononetin did not significantly affect apoptosis. In the wound healing assay, formononetin treatment revealed a more significant decrease in the proliferation of the eutopic endometrium in patients with endometriosis than in the eutopic endometrium without endometriosis. Relative expression of sex hormone receptors decreased with increasing formononetin doses. Although no significant differences were observed in the ER, PR-A, ERβ/ERα, and PR-B/PR-A, significant down-regulation of PR-B expression was noted after formononetin treatment at 80 μM. In the in vivo study, endometriotic lesions in the formononetin-treated group significantly decreased compared to those in the control group. The relative expression of p27 using IHC was highest in the control group and lowest in the high-dose formononetin treatment group. Conclusions: Formononetin treatment was shown to inhibit the proliferation of eutopic and ectopic endometria in patients with endometriosis through the regulation of p27, pSTAT3, and PR-B. In an endometriosis mouse model, formononetin treatment significantly reduced the number of endometriotic lesions with decreased p27 expression. The results of this study suggest that formononetin may be used as a non-hormonal treatment option for endometriosis.