| Embryo Implantation is the process that mother and embryo recognize each other. In this specific period (5-6 days after fertilization), the specific parts of the maternal endometrium (uterus or uterine wall than anterior wall of the lower part) show "acceptance" while the blastocyst has "immersed in nature." This process comprises four steps: blastocyst adheres in the intima; trophoblast epitaxial grows and forms neointimal gap; blastocyst invades the endometrium; fertilization, endometrial epithelial hyperplasia closes the last gap, blastocyst is completely buried in the endometrium.Pyrogallic Acid is an organic acid widely present in Shidi, grapes, tea and other plants. Studies have shown that Pyrogallic Acid has significant anti-fertility, anti-tumor, anti-oxidation anti-virus and many other biological effects. In the rabbit anti-fertility screening, primary results indicate Shidi containing Pyrogallic acid has some anti-fertility effect. But so far, reports about Pyrogallic Acid's anti-fertility effects and its mechanism have not been found. In our study, we aim to observe anti-fertility effect of Pyrogallic Acid in female mice, and further investigate its anti-fertility mechanism from the aspects of endometrial receptivity, sex hormone levels, cytokines, MMPs expression and so on.Materials and Methods: The groups were divided into the blank gel (matrix) control group (given blank gel by vaginal injection), three doses vaginal administration groups (10 mg·kg-1, 30mg·kg-1, 100mg·kg-1 Pyrogallic Acid respectively), intraperitoneal injection group(100 mg·kg-1 Pyrogallic acid by intraperitoneal injection) and positive control group(administered mifepristone). Sexually mature ICR male mice and female mice were caged together at ratio of 3:1 for 12h and the female vaginal suppository was checked the next morning, mice with pessary were thought as pregnancy day 1 (D1). Female mice with pessary were randomly divided into 6 groups, 10 for each group. The pregnant mice number, the fetal number and pregnancy rate were calculated. Mice ovaries, uterus from each group were obtained. By using Immunohistochemical analysis, we detected MMP-2, MMP-9, TGF-β, ICAM-1 andβ-catenin expression levels in endometrial tissue; by real-time quantitative PCR technique, we investigated ICAM-1 andβ-catenin transcriptional levels in endometrial tissue during pregnancy and the transcriptional regulation of Pyrogallic acid on ICAM-1 andβ-catenin expression. By ELISA we observed the effect of Pyrogallic acid on IGFBP1 and LIF expression levels in endometrial cells. In vitro experiments, we used cultured endometrial cells, different concentrations of Pyrogallic Acid were administered when 80% cells were fused, the cells were collected 24h after for gelatin zymography analysis and implantation of mouse blastocysts in vitro, we analyzed window period fertility factor IGFBP1 and LIF levels in endometrial cell supernatant.Results: (1) Pyrogallic Acid by vaginal injection can reduce female pregnancy rate and fetal number to some extent. The implantation rates in female mice were significantly reduced both in100 mg·kg-1 vaginal delivery group and intraperitoneal injection group. Pyrogallic acid enabled endometrial changes in organizational structure and endometrial hyperplasia and thickening were significantly weaker than those in the control group. Moreover, angiogenesis was also not obvious.(2) With cultured endometrial cell we did experiment in implantation mouse blastocysts, results showed that Pyrogallic acid groups, blastocyst adhesion rate was decreased in low-dose group, but compared with the control group it was not statistically significant. However, in 50 mol·L-1 and 100 mol·L-1 groups, blastocyst adhesion rates were decreased significantly compared with the control group. Blastocyst expansion rate of 25 mol·L-1 Pyrogallic acid group is 33.33%, while in 50 mol·L-1-dose group, blastocyst expansion rate is 28.89%, 100 mol·L-1 extended-dose group was 31.85% . There were no significant difference between different dose groups, but difference exists when compared with the control group.(3) The endometrial cell was co-cultured, different concentrations of Pyrogallic Acid were given when the cells were reached to 80% confluence. 24h later the cells were collected for gelatin zymography analysis. We found that treated cells with 50 mol·L-1 and 100 mol·L-1of Pyrogallic acid, MMP-2 and MMP-9 activities were significantly decreased. In vivo results show that MMP-2 and MMP-9 protein in normal 4d pregnant mice endometrium is moderate expressed, however, Pyrogallic acid decreased MMP-2 and MMP-9 expression.(4) By real-time quantitative PCR technique, we investigated ICAM-1 andβ-catenin transcriptional levels in endometrial tissue during pregnancy and the transcriptional regulation of Pyrogallic Acid on ICAM-1 andβ-catenin expression. The results showed that, compared with the control group, the ICAM-1levels in mice endometrial tissue decreased by 1.28,2.67 and 3.2-fold after treated with 25 mol·L-1, 50 mol·L-1 and 100 mol·L-1 of Pyrogallic acid respectively, whileβ-catenin transcriptional levels decreased by 1.44, 2.17 and 2.97 times.(5) By using ELISA analysis we investigated IGFBP1 and LIF levels in endometrial cell culture supernatant to observe effect of Pyrogallic acid on the endometrial cells IGFBP1 and LIF exression. Results demonstrated positive drugs and Pyrogallic acid can both inhibit endometrial cells IGFBP1 and LIF. 50 mol·L-1 and 100 mol·L-1 of Pyrogallic acid can significantly reduce endometrial cells TGF-β, IGFBP1 and LIF (P <0.05), with statistically significant difference.Conclusion:First, Pyrogallic Acid can reduce fertility in female mice and have effects of anti-implantation, anti-early pregnancy. Pyrogallic acid can change the organizational structure of mouse endometrium and inhibit endometrial cell proliferation and angiogenesis.Second, 50 mol·L-1 and 100 mol·L-1 Pyrogallic acid can reduce blastocyst adhesion rate and expansion rate in vitro.Third, in the normal endometrial cells, MMP-2 and MMP-9 was the most active, MMP-2 and MMP-9 activities in positive control group decreased, 50 mol·L-1 and 100 mol·L-1 of Pyrogallic acid , MMP-2 and MMP-9 activities were significantly decreased. Therefore, Pyrogallic Acid may have impact on endometrial receptivity by influencing MMP-2 and MMP-9 activities. Four, 25 mol·L-1, 50 mol·L-1 and 100 mol·L-1 of Pyrogallic Acid can decrease the mouse endometrium of ICAM-1 andβ-catenin transcriptional level compared with control group, suggesting that Pyrogallic acid can affect endometrial receptivity by regulating the ICAM-1 andβ-catenin transcription.Fifth, in the endometrial tissue and cells, 50 mol·L-1 and 100 mol·L-1 -dose Pyrogallic Acid group were significantly reduced endometrial cells IGFBP1 and LIF. It indicated that Pyrogallic Acid can reduce the implantation window endometrial receptivity to produce anti-fertility effect.Innovation: The anti-fertility effect of Pyrogallic Acid is mainly reflected in the inhibition of embryo implantation, regulation of endometrial MMP-2 and MMP-9 expression and affecting the degradation process of stromal cells. At the same time Pyrogallic Acid can inhibit the adhesion molecules ICAM-1 andβ-catenin expression in endometrial tissue and affect the adhesion of the blastocyst. Pyrogallic Acid can also change endometrial cells secreted TGF-β, IGFBP1, and LIF. Anti-fertility effect of Pyrogallic Acid was achieved by multi-level interference endometrial receptivity and inhibition endometrial adhesion and expansion of the blastocyst.
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