The Effect And Molecular Mechanism Of AIF-1 On Endometrium With GnRH Antagonist Protocol

Studies have shown that GnRH antagonist protocol cycles have lower implantation and clinical pregnancy rates than GnRH agonist long protocol cycles.Endometrial receptivity is a key factor contributing to this phenomenon;however,the mechanism is still unknown.Many studies indicated that aberrant inflammation may account for this situation.Allograft inflammatory factor-1(AIF-1)is a cytokine associated with inflammation and allograft rejection.Microarray results revealed that AIF-1 was upregulated in the antagonist group compared with the other two groups.However,the effect of AIF-1 on endometrial receptivity hasn’t been reported.In this study,number of available embryo,implantation and pregnancy rates were studied in 238 patients undergoing their first cycle of IVF/ICSI between 2012 and 2014.Forty of these patients opted to have no fresh embryo replacement and were divided into two equal groups:(i)GnRH antagonist protocol and(ii)GnRH agonist long protocol,group 3 included 20 infertile women with a tubal factor in untreated cycles.Endometrial tissues from mid-secretory phase(implantation phase)were got from these patients after assining informed consent.During the same interval,endometrial tissues were taken from 18 infertile women with a tubal factor in the early proliferative phase,late proliferative phase,and mid-secretory phase of the menstrual cycle(n = 6/group).Microarray analysis,q PCR,Western blot analysis and immunohistochemistry were used to investigate the expression levels of AIF-1 and the related cytokines(TNF-α,IL1β,IL1 RA,IL6,IL12,IL15 and IL18).The effect of AIF-1on uterine receptivity was modeled using in vitro adhesion experiments(coculture of JAR cells and Ishikawa cells)and the proliferation assay.Analysis of clinical data of 238 patients found that compared with the GnRH agonist group,the implantation rate and pregnancy rate of the GnRH antagonist group decreased,but the number of available embryos was not significantly different,suggesting that the patients using GnRH antagonist were experiencing impaired endometrial receptivity.From the in vitro experiment,we found that the expression of AIF-1 was the highest in early proliferative phase,decreasing thereafter in the late proliferative phase,and almost disappearing in the mid-secretory phase,indicating that low AIF-1 expression might be important for embryo implantation during implantation phase.Microarray results revealed that AIF-1 was upregulated in the antagonist group compared with the control group(fold change [FC] =3.75)and the agonist(FC = 2.20)group.And the following validation with expanded samples indicated that both the m RNA and protein expression levels of AIF-1 and TNF-αwere the higher in the antagonist group than in the other two groups(P < 0.05)which did not differ significantly(P > 0.05).The protein levels of TNF-α in both Ishikawa cells and primary endometrial cells were significantly increased(P < 0.05)at 96 h after transfection with the AIF-1 expression vector,indicating that TNF-α was mediated by AIF-1 in endometrial cells.Overexpression of AIF-1 in Ishikawa cells inhibited adhesion of JAR cells to them.However,there is no change on the proliferation of Ishikawa cells following AIF-1 overexpression.Thus,increased AIF-1 might inhibit adhesion during implantation via raised TNF-α.Our findings indicated AIF-1 is increased in IVF cycles with GnRH antagonist protocol and mediates greater TNF-α expression during implantation phase,which may be unfavorable for embryo implantation.