| Objective:To explore the role of Kisspeptin in the regulation of endometrial decidualization and its role in the pathogenesis of abortion.Methods:1.During March 2018 to October 2018,21 patients with missed abortion and 13 patients with post-abortion non-pregnant women and 24 normal early pregnancy women and 15 normal non-pregnant women were selected from the Second Affiliated Hospital of Soochow University.Immunosorbent assays were used to detect the expression of Kisspeptin in serum.2.Cell experiments:Endometrial stromal cells were cultured in vitro,and decidualization was induced for 96 h.(1)Inducing decidualization of endometrial stromal cells,and observing the morphological changes of cells by light microscopy and HE staining.(2)Cell grouping:Kiss-1 siRNA group;different concentrations of Kisspeptin intervention group;Kiss-1 siRNA+exogenous Kisspeptin group;non-intervention group.The decidualization of endometrial stromal cells was induced.After 24h,48h,72h,96h and 120h,immunofluorescence staining of insulin-like growth factor binding protein-1(IGFBP-1)and prolactin(PRL)were performed.Western-blot detects PRL and progesterone,RT-qPCR detects progesterone rate-limiting enzymes(StAR,P450scc).Results:1.Serum Kisspeptin was extremely low in non-pregnant patients,but was significantly elevated in pregnant women(P<0.0001).Serum Kisspeptin in patients with missed abortion was significantly lower than normal pregnancy(P<0.0001),but there was no significant difference between the two non-pregnancy groups(P>0.05).2.Under light microscopy and HE staining,the surface area of endometrial stromal cells gradually increased(P<0.0001)and cell roundness increased(P<0.001).The decidualization markers IGFBP-1 and PRL gradually increased.3.Immunofluorescence showed that IGFBP-1 and PRL were mainly localized in the nucleus and cytoplasm and expressed in the cell.The expression of decidualization markers was down-regulated by siRNA inhibition of Kiss-1 expression(P<0.0001).After over-expression of 250nM,500nM,1μM Kisspeptin or Kiss-1,the expression of decidualization markers was stronger than that of normal decidual cells(P<0.0001),but there was no significant difference between different concentrations(P>0.05).Western Blot assay showed that the expression of IGFBP-1 and PRL increased gradually with the decidualization time(P<0.0001).The expression of IGFBP-1 was down-regulated after normalization of Kisspeptin compared with normal decidual cells(P<0.01).IGFBP-1 was elevated after addition of 1μM Kisspeptin,which was similar to that of normal decidual cells(P>0.05).The expression of IGFBP-1 was significantly higher than that of normal decidual cells(P<0.001),but the expression of IGFBP-1 was not significantly different in Kisspeptin concentration(P>0.05).4.The results of RT-qPCR showed that the expression of progesterone rate-limiting enzyme(STAR,P450scc)was increased in the endometrial stromal cells after decidualization.Inhibition of KISS-1 gene significantly increased progesterone rate-limiting enzyme expression(P<0.0001),StAR expression was increased after exogenous addition of Kisspeptin(P<0.0001),while P450scc expression decreased(P<0.05)..The expression of Kisspeptin using StAR expression(P<0.01)and P450scc(P<0.05)was lower than that of normal decidual cells.The expression of prostaglandin rate-limiting enzyme was not significantly affected by different Kisspeptin concentrations(P>0.05).Western Blot The progesterone concentration increased with the decidualization process(P<0.0001).The progesterone expression after Kisspeptin inhibition was lower than that of exogenous Kisspeptin(P<0.05),and the progesterone expression increased with the increase of Kisspeptin concentration(P<0.001).Conclusions:1.The decrease in serum Kisspeptin expression in patients with missed abortion suggests that Kisspeptin is expected to be promising biomarker of pregnancy viability,and may have clinical utility in developing an accurate test of early pregnancy outcome in the future.2.Kisspeptin is positively correlated with decidualization level.Kisspeptin can regulate decidualization of the uterus.There may be a synergistic compensation mechanism between Kisspeptin and progesterone,which needs further research and verification. |
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