Increased AIF-1-mediated TNF-α expression during implantation phase in IVF cycles with GnRH antagonist protocol

Abstract

Study question: Is allograft inflammatory factor-1 (AIF-1), a cytokine associated with inflammation and allograft rejection, aberrantly elevated in in vitro fertilization (IVF) cycles with gonadotropin-releasing hormone (GnRH) antagonist protocol with potential effects on endometrial receptivity?

Summary answer: 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.

What is known already: Studies have shown that GnRH antagonist protocol cycles have lower implantation and clinical pregnancy rates than GnRH agonist long protocol cycles. Endometrial receptivity but not embryo quality is a key factor contributing to this phenomenon; however, the mechanism is still unknown.

Study design, size, duration: 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. 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).

Participants/materials, setting, methods: Microarray analysis, RT-qPCR, Western blot analysis, immunohistochemistry were used to investigate the expression levels of AIF-1 and the related cytokines (TNF-α, IL1β, IL1RA, IL6, IL12, IL15 and IL18). The effect of AIF-1 on uterine receptivity was modeled using in vitro adhesion experiments (coculture of JAR cells and Ishikawa cells).

Main results and the role of chance: 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. The raw microarray data and complete gene expression table were uploaded to GEO under the accession number of GSE107914. Both the mRNA and protein expression levels of AIF-1 and TNF-α were 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. Thus, increased AIF-1 might inhibit adhesion during implantation via raised TNF-α.

Limitations reasons for caution: The sample size of the microarray was small, which might weaken the accuracy of our results; however, the sample size of RT-qPCR and the Western blotting assays were sufficient to compensate for this deficiency in our study. In addition, the aberrant AIF-1 and thus TNF-α expression is one of many factors that may contribute to limiting implantation success. Therefore, further extensive in vitro mechanistic and in vivo animal studies are needed to assess the actual functional impact of this pathway.

Wider implications of the findings: Anti-TNF-α therapy might mitigate the adverse effects of GnRH antagonist on endometrial receptivity and improve the implantation rate in GnRH antagonist protocols in IVF.

Study funding/competing interests: This work was supported by grants from the National Natural Science Foundation of China, Grant numbers 81771656 and 81370763; Clinical research special fund of Chinese Medical Association, Grant number 16020480664; Shanghai Jiao Tong University Medicine-Engineering Fund, Grant number YG2017ZD11 and YG2017MS57; and the Merck-Serono China Research Fund for Fertility Agreement. P.C.K.L. is supported by a Canadian Institutes of Health Research Foundation Scheme Grant 143317. None of the authors has any competing interests.

Figure 1

Representative images and quantification of AIF-1 in endometrial tissue from fertile women at different phases in the menstrual cycle (n = 6 per group) using Western blotting. All data are presented as mean values ± SEM. EP, early proliferative phase; LP, late proliferative phase; MS, mid-secretory phase. The differences between groups were analyzed by Kruskal–Wallis test, followed by multiple comparisons using Mann–Whitney U-test. Data were considered statistically significant if P < 0.05.

Figure 2

AIF-1 microarray expression and validation (n = 3 per group). (A and B) Functional enrichment results of differentially expressed genes (DEGs), including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO). P-values and gene count of each term are shown as bar plots and lines, respectively. Red text indicates that AIF-1 was among the genes identified. (C) Protein and protein interaction network. Red nodes indicate upregulated proteins, and green nodes indicate downregulated proteins. The size of the node was positively correlated with the quantity of related genes and the larger module was associated with immune activation and contained AIF-1 (red arrow). (D) AIF-1 mRNA expression between the control, GnRH agonist (GnRH ago) and GnRH antagonist (GnRH ant) groups using microarray. ‘FC’ refers to fold change. The y-axis is log 2 scaled and the upper whisker, upper side of box, thick line in the box, lower side of box and the lower whisker corresponds to maximum value, upper 75% quantile, median, lower 25% quantile and minimum value, respectively. (E) AIF-1 mRNA expression in the GnRH agonist/control, GnRH ant/control, and GnRH ant/GnRH agonist using microarray and RT-qPCR analyses. Error bars are expressed as SEM.

Figure 3

AIF-1 expression levels in the control group, GnRH agonist group and GnRH antagonist group. (A) Graphs illustrate the relative mRNA level of AIF-1 among the three groups as assayed by RT-qPCR. Analyzed by ANOVA, followed by Bonferroni test. (B) Representative Western blotting images and quantification of AIF-1 protein in the three groups. (C) Representative images and quantification of AIF-1 in the three groups as assessed by immunohistochemistry. All data are presented as mean ± SEM. The differences between groups were analyzed by Kruskal–Wallis test, followed by multiple comparisons using Mann–Whitney U-test in panel B and C. Data were considered statistically significant if P < 0.05.

Figure 4

Changes in cytokine levels following force-expression of AIF-1. (A) TNF-α, IL1β, IL1RA, IL6, IL12, IL15 and IL18 mRNA levels assayed by RT-qPCR at 0, 24, 48, 72 and 96 h after transfection with AIF-1-expression vector. All the cytokines increased significantly (P < 0.05 or P < 0.01), except for IL6. (B) Levels of TNF-α, IL1β, IL1RA, IL12, IL15 and IL18 mRNA after 72 h of transfection with AIF-1-expression vector (0.5 or 1 μg). TNF-α, IL1β and IL1RA mRNA levels were significantly higher than the control (P < 0.01). (C) Changes in TNF-α, IL1β and IL1RA protein levels analyzed by Western blotting at 96 h after transfection with the AIF-1-expression plasmid in Ishikawa cells. Only TNF-α was significantly increased (P < 0.05). (D) Changes of TNF-α protein level analyzed by Western blotting at 96 h after transfection with the AIF-1-expression plasmid in primary endothelial and stromal cells. TNF-α was significantly increased after AIF-1 plasmid transfection (P < 0.05). All data are presented as the mean ± SEM. The differences between groups of all data were analyzed by ANOVA, followed by multiple comparisons using Bonferroni test (except AIF-1 expression in Figure 4B, which was analyzed by Kruskal–Wallis test, followed by multiple comparisons using Mann–Whitney U-test). Data were considered statistically significant if P < 0.05.

Figure 5

The expression of cytokines in the control, GnRH agonist, and GnRH antagonist groups. (A) Graphs illustrate the relative mRNA levels of TNF-α, IL1RA, IL1 β, IL6, IL12, IL15, IL18 among the three groups as assayed by RT-qPCR. (B) Representative western blotting images and quantification of TNF-α protein in the three groups. (C) Representative images and quantification of TNF-α in the three groups as assessed by immunohistochemistry. All data are presented as mean ± SEM. The differences between groups of TNF-α and IL18 were analyzed by Kruskal–Wallis test, followed by multiple comparisons using Mann–Whitney U-test and the differences between groups of IL1RA, IL1 β, IL6, IL12, IL15 were analyzed by ANOVA. Data were considered statistically significant at P < 0.05.

Figure 6

Effect of AIF-1 on the adhesion of JAR cells to Ishikawa cells. Ishikawa cells were treated with negative or AIF-1 expression lentivirus. Fluorescence-labeled JAR cells were calculated and expressed as a fold of negative group. All data are presented as mean ± SEM. The differences between groups were analyzed by independent sample T-test. Data were considered statistically significant if P < 0.05.