Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells

Abstract

Background: Successful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells' ability to invade the endometrium. We hypothesized that PIF's effects on the endometrium counteract its pro-invasive effects.

Methods: We tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay.

Results: Firstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line's invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF's anti-invasive action was associated with a specifically decrease in MMP-9 activity.

Conclusion: Taken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Keywords: Decidualization; Embryo implantation; Human endometrium; Preimplantation factor (PIF); Signal transduction; Trophoblastic invasion.

Fig. 1

Effect of sPIF on human ESC decidualization. Human ESCs were cultured in DMEM/F12 medium supplemented with E2, P4 and (in some experiments) 50 nM sPIF for 15 days. A-C Total RNA was extracted after three (D3), eight (D8), and 15 days (D15) of cell decidualization. mRNA expression levels of prolactin (A), IGFBP-1 (B), and connexin-43 (C) were quantified by RT-qPCR, as described in the Materials and Methods section. The data are presented as the mean ± SEM of 6 to 10 separate experiments. D Prolactin secretion into the endometrial supernatants was measured after 3 days (D3), 8 days (D8), and 15 days (D15) of cell differentiation, as described in the Materials and Methods. The data are presented as the mean ± SEM of 8 to 13 separate experiments. The control values on D3, D5, D8, D11, D13, and D15 were 46.5 ± 8.7, 130.7 ± 22.5, 346 ± 110, 419 ± 105, 494.5 ± 77 and 676.4 ± 102 mIU/g protein, respectively. *: P < 0.05; **: P < 0.01; ***: P < 0.001 (Wilcoxon test); (a) Prolactin secretion in presence of sPIF vs. the control (lacking sPIF); (b) Prolactin secretion at D15 vs. D3 for control experimental condition (lacking sPIF)

Fig. 2

Effect of a PI3K inhibitor on sPIF-enhanced ESC decidualization. Human ESCs were cultured in DMEM/F12 medium supplemented with E2, P4, and the PI3K inhibitor wortmannin (10 μM) in the presence or in the absence of sPIF (50 nM) for 15 days. A-B Total RNA was extracted after 15 days (D15) of cell decidualization. mRNA expression levels of IGFBP-1 (A), and connexin-43 (B) were quantified by RT-qPCR, as described in the Materials and Methods section. The data are presented as the mean ± SEM of 4 to 11 separate experiments. *: P < 0.05; **: P < 0.005 (Wilcoxon test). (a) sPIF vs. control (lacking sPIF); (b) wortmannin vs. control (lacking sPIF); (c) sPIF vs. sPIF + wortmannin

Fig. 3

Effect of sPIF on human ESC motility. Human ESCs were cultured in DMEM/F12 medium supplemented with E2, P4 and (in some experiments) 50 nM sPIF for 15 days. Wound width was analyzed 0 and 24 h after wounding, as described in the Materials and Methods. The wound width data are expressed as the mean ± SEM of 5 separate experiments. *: P < 0.05 (Wilcoxon test) for sPIF vs. the control (lacking sPIF)

Fig. 4

Involvement of sPIF in the endometrial control of trophoblast invasion by ESCs. PIF improves the endometrial control of trophoblastic migration. A Transwell migration assays of HTR-8/SVneo cells were performed as described in the Materials and Methods. B Representative microphotographs of 12 separate experiments showing fixed and DAPI stained HTR-8/SVneo cells on the bottom side of the transwell membrane. HTR-8/SVneo cells were cultivated with conditioned medium (CM) from decidualized ESCs exposed for 15 days in control condition (a) or with 50 nM sPIF (b). C HTR-8/SVneo cells were suspended in the presence of CM from decidualized ESCs having been exposed for 15 days in presence or absence of sPIF (50 nM) or in the presence of control medium, supplemented or not with FCS 10% or adiponectin (250 ng/ml). The data are presented as the mean ± SEM of 3–12 separate experiments. ***: P < 0.001 (Wilcoxon test) for sPIF vs. the control (lacking sPIF)

Fig. 5

Effect of sPIF on endometrial TIMP mRNA expression and MMP activities. Human ESCs were cultured in DMEM/F12 medium supplemented with E2, P4 and (in some experiments) 50 nM sPIF for 15 days. A Activities of gelatinases (MMP-9 and MMP-2) in CM from decidualized ESCs having been exposed for 15 days in presence or absence of sPIF (50 nM), as described in the Materials and Methods. This figure shows one representative of 11 separate experiments. B Quantification of gelatin zymography results for MMP-2 and MMP-9 activities. The data are presented as the mean ± SEM of 5 to 8 separate experiments. C Total RNA was extracted after 15 days of cell differentiation. mRNA expression levels of TIMP-1 and TIMP-2 were quantified by RT-qPCR, as described in the Materials and Methods. The data are presented as the mean ± SEM of 11 separate experiments. **: P < 0.005 (Wilcoxon test) for sPIF vs. the control (lacking sPIF)