Figure 1 illustrates the expression patterns and diagnostic relevance of NCOA4 in both ectopic and eutopic endometrial tissues in EMS patients, utilizing data from GSE51981 and GSE58178 in the training set. The results demonstrate a notable decrease in NCOA4 levels in the eutopic endometrial tissues of EMS patients relative to those in the control group. (P < 0.001, Fig. 1A). The AUROC for diagnosing eutopic endometrium in EMS was determined to be 0.68 (95% CI: 0.77 − 0.59, Fig. 1B). In contrast, NCOA4 was significantly upregulated in the ectopic endometrial tissues of EMS patients (P < 0.05, Fig. 1C), with an AUROC for diagnosing ectopic endometrium in EMS at 0.83 (95% CI: 1.0-0.57, Fig. 1D).
3.2 Enhanced Expression of NCOA4 in Eutopic and Ectopic Endometrium of EMS in the Validation Set
Figure 2 highlights the expression and diagnostic significance of NCOA4 in both eutopic and ectopic endometrial tissues of EMS in the validation datasetResults demonstrated a notable upregulation of NCOA4 in eutopic endometrial tissues of EMS patients compared to the control group (P < 0.0001, Fig. 2A). The AUROC for diagnosing eutopic endometrium in EMS was 0.77 (95% CI: 0.84 − 0.69, Fig. 2B). Similarly, a significant upregulation of NCOA4 was observed in ectopic endometrial tissues of EMS patients (P < 0.01, Fig. 2C), with an AUROC for diagnosing ectopic endometrium in EMS at 0.66 (95% CI: 0.75 − 0.57, Fig. 2D).
3.3 Differential Expression of Various Immune Cell Types and Aberrant Overexpression of Immune Checkpoint Genes in Eutopic and Ectopic Endometrium of EMS Patients
The CIBERSORT algorithm was utilized to evaluate the variations in immune cells between eutopic and ectopic endometrial tissues of EMS patients in comparison to control eutopic endometrium. Figure 3A illustrates the proportionate expression of 22 immune cell types in the eutopic endometrium of EMS patients versus the control eutopic endometrium, with the initial 77 cases representing EMS patient eutopic endometrium and the subsequent 71 cases representing control eutopic endometrium. Figure 3B reveals significant differential expression in six immune cell types in eutopic endometrium of EMS patients compared to controls. Three cell types (Plasma cells, T cells CD8, NK cells resting) were more prevalent, while three types (T cells gamma delta, Macrophages M2, Dendritic cells activated) were less expressed in the eutopic endometrium of EMS patients. Figure 3C displays the proportionate expression of 22 immune cell types in ectopic endometrium of EMS patients versus control eutopic endometrium, with the first 149 cases being ectopic endometrium of EMS patients and the last 43 being control eutopic endometrium. Figure 3D indicates that 15 immune cell types demonstrated significant variance in expression in ectopic endometrium of EMS patients compared to controls, with eight types (B cells Memory, T cells CD4 memory resting/activated, Monocytes, Macrophages M1/M2, Mast cells resting, Neutrophils) exhibiting increased expression, and seven types (Plasma cells, T cells CD8, T cells follicular helper, Tregs, NK cells resting/activated, Dendritic cells activated) showing decreased expression.
Further analysis of microarray data was conducted to explore the disparities in immune checkpoint gene expression between eutopic and ectopic endometrial tissues of EMS patients compared to the control group. The findings indicated that, relative to the control group, five out of eight immune checkpoint genes exhibited significant overexpression in the eutopic endometrium of EMS patients (Fig. 3E), including CTLA4, LAG3, CD48, CD27, CD40. In the ectopic endometrium of EMS patients, six out of eight genes were significantly overexpressed (Fig. 3F), comprising CTLA4, IDO1, LAG3, CD48, CD27, CD40. In summary, immune checkpoint genes showed anomalously high expression in both eutopic and ectopic endometrial tissues of EMS patients.
3.4 Correlation Between the Differential Expression of Key Iron Autophagy Genes NCOA4, FTH1, SQSTM1 (P62), MAP1LC3B (LC3B) and Immune Cells, Immune Checkpoint Genes in Ectopic and Eutopic Endometrium of Patients with EMS
Figure 4A displays the correlation between four crucial iron autophagy genes (NCOA4, FTH1, SQSTM1(P62), MAP1LC3B(LC3B)) and immune cells in eutopic endometrium of EMS patients. NCOA4 exhibited significant correlations with 10 immune cell types, showing positive correlations with six and negative correlations with four, with the strongest correlations being with NK cells resting (r = -0.64, P < 0.001) and T cells gamma delta (r = 0.54, P < 0.001). FTH1 correlated significantly with six immune cell types, predominantly negative (4/6), with the strongest being with Plasma cells (r = -0.35, P < 0.01). SQSTM1(P62) correlated significantly with 11 immune cell types, positively with five and negatively with six, with the strongest correlations being with NK cells resting (r = 0.58, P < 0.001) and T cells gamma delta (r = -0.59, P < 0.001). MAP1LC3B(LC3B) showed significant correlations with six immune cell types, positively with three and negatively with three, with the strongest correlations being with T cells CD8 (r = -0.52, P < 0.01) and NK cells activated (r = 0.47, P < 0.01).
Figure 4B illustrates the correlation between these key iron autophagy genes and immune cells in ectopic endometrium of EMS patients. NCOA4 showed negative correlations with four immune cell types, the strongest being with T cells CD4 memory activated (r = -0.2, P < 0.05). FTH1 negatively correlated with three immune cell types, with the strongest being with B cells memory (r = -0.22, P < 0.01). SQSTM1(P62) displayed no notable correlation with immune cells. MAP1LC3B(LC3B) was significantly correlated with three immune cell types, mostly negative (2/3), with the strongest being with B cells memory (r = -0.2, P < 0.05).
In eutopic endometrium of EMS patients, the correlation of four key iron autophagy genes with immune checkpoint genes is shown in Fig. 4C. NCOA4 negatively correlated with CTLA4 (r = -0.25, P < 0.05), LMTK3 (r = -0.45, P < 0.001), LAG3 (r = -0.36, P < 0.01), CD48 (r = -0.24, P < 0.05), CD27 (r = -0.79, P < 0.0001), CD40 (r = -0.53, P < 0.001), and positively with IDO1 (r = 0.38, P < 0.01). FTH1 positively correlated with CD27 (r = 0.29, P < 0.05), CD40 (r = 0.25, P < 0.05). SQSTM1(P62) showed positive correlations with LMTK3 (r = 0.47, P < 0.001), LAG3 (r = 0.36, P < 0.01), CD48 (r = 0.26, P < 0.05), CD27 (r = 0.71, P < 0.0001), CD40 (r = 0.62, P < 0.001), and a negative correlation with IDO1 (r = -0.33, P < 0.01). MAP1LC3B(LC3B) negatively correlated with CTLA4 (r = -0.26, P < 0.05), CD27 (r = -0.44, P < 0.001), and positively with IDO1 (r = 0.76, P < 0.0001).
In ectopic endometrium of EMS patients, the correlation of these genes with immune checkpoint genes is depicted in Fig. 4D. NCOA4 positively correlated with CTLA4 (r = 0.46, P < 0.001), IDO1 (r = 0.41, P < 0.001), LMTK3 (r = 0.73, P < 0.0001), LAG3 (r = 0.36, P < 0.001), CD48 (r = 0.66, P < 0.0001), CD27 (r = 0.42, P < 0.001), CD40 (r = 0.22, P < 0.01), and negatively with ICOS (r = -0.48, P < 0.001). FTH1 positively correlated with CTLA4 (r = 0.44, P < 0.001), IDO1 (r = 0.35, P < 0.001), LMTK3 (r = 0.67, P < 0.0001), LAG3 (r = 0.34, P < 0.001), CD48 (r = 0.72, P < 0.0001), CD27 (r = 0.37, P < 0.001), CD40 (r = 0.16, P < 0.05), and negatively with ICOS (r = -0.48, P < 0.001). SQSTM1(P62) showed positive correlations with CTLA4 (r = 0.33, P < 0.001), IDO1 (r = 0.79, P < 0.0001), LMTK3 (r = 0.75, P < 0.0001), LAG3 (r = 0.55, P < 0.0001), CD48 (r = 0.51, P < 0.0001), CD27 (r = 0.80, P < 0.0001), CD40 (r = 0.56, P < 0.0001), and MAP1LC3B(LC3B) positively correlated with CTLA4 (r = 0.46, P < 0.001), IDO1 (r = 0.56, P < 0.0001), LMTK3 (r = 0.76, P < 0.0001), LAG3 (r = 0.39, P < 0.001), CD48 (r = 0.67, P < 0.0001), CD27 (r = 0.58, P < 0.001), CD40 (r = 0.34, P < 0.001), and negatively with ICOS (r = -0.29, P < 0.001).
In summary, in eutopic endometrial tissues of EMS patients, NCOA4 showed a notable positive association with one immune checkpoint gene and negative associations with six others; moreover, FTH1 showed positive correlations with two immune checkpoint genes; SQSTM1(P62) positively with five and negatively with one; and MAP1LC3B(LC3B) positively with one and negatively with two immune checkpoint genes. In ectopic endometrial tissues, NCOA4 showed a positive correlation with seven immune checkpoint genes, and a negative correlation with one; FTH1 positively with seven and negatively with one; SQSTM1(P62) positively with all seven; and MAP1LC3B(LC3B) positively with seven and negatively with one.
The investigation of free iron content in the eutopic endometrium of the control group and in both eutopic and ectopic endometrium of EMS rats indicated a significant elevation in free iron content in the ectopic endometrium, while a noticeable reduction was observed in the eutopic endometrium of EMS rats compared to the control group. These findings imply disruptions in iron metabolism within eutopic as well as ectopic endometrial tissues in rats of EMS (refer to Table 2).
Table 2
Quantification of Free Iron in Endometrial Tissues of Various Rat Groups (n = 3, mean ± SD)
Group
|
Healthy Rat Endometrium
|
EMS Rat Eutopic Endometrium
|
EMS Rat Ectopic Endometrium
|
Free Iron Content (µmol/kg)
|
386.75 ± 42.6
|
165.3 ± 7.63###
|
756.01 ± 51.79###
|
Note: Data are presented to two decimal places. Compared to the healthy rat endometrium, ###P < 0.001. |
3.7 Altered Protein Levels of NCOA4, LC3B, FTH1, and P62 in Eutopic and Ectopic Endometrium of EMS Rats
Iron autophagy, facilitated by NCOA4, involves the degradation of cellular ferritin FTH1 in autolysosomes, thus releasing free iron ions, pivotal in iron metabolism under both physiological and pathological states. To investigate the cause behind abnormal free iron levels in eutopic and ectopic endometrial tissues of EMS rats, the expression levels of iron autophagy-associated proteins such as NCOA4, LC3B, FTH1, and P62 were examined.. Compared to the control group, ectopic endometrial tissues in EMS rats exhibited increased expression of NCOA4 and LC3B proteins (P < 0.01) and decreased expression of FTH1 and P62 proteins (P < 0.05). In contrast, eutopic endometrial tissues showed decreased expression of NCOA4 and LC3B proteins (P < 0.01 or 0.001) and increased expression of FTH1 and P62 proteins (P < 0.01), as illustrated by Fig. 6 and detailed within Table 3.
Table 3
Relative Expression Levels of NCOA4, LC3B II/I, FTH1, and P62 Proteins in Healthy, Eutopic, and Ectopic Endometrium of Rats (x¯±s)
Group
|
Number
|
NCOA4
|
LC3BⅡ/Ⅰ
|
FTH1
|
P62
|
Ectopic Endometrium (EMS)
|
4
|
1.05 ± 0.05**
|
4.17 ± 2.11**
|
0.44 ± 0.31*
|
0.19 ± 0.31*
|
Healthy Endometrium
|
4
|
0.75 ± 0.19
|
1 ± 0.26
|
0.86 ± 0.07
|
0.75 ± 0.11
|
Eutopic Endometrium (EMS)
|
4
|
0.2 ± 0.18***
|
0.36 ± 0.33**
|
1.13 ± 0.07**
|
1.02 ± 0.07**
|
Note: Data are presented to two decimal places. Compared to healthy endometrium, ***P < 0.001, **P < 0.01, *P < 0.05. |
3.8 mRNA Expression Variations of NCOA4, LC3B, FTH1, and P62 in Eutopic and Ectopic Endometrium of EMS Rats
Further analysis of mRNA expression for iron autophagy-related proteins NCOA4, LC3B, FTH1, and P62 was conducted in eutopic and ectopic endometrial tissues of EMS rats. Compared to healthy rat endometrium, ectopic endometrial tissues of EMS rats displayed an upward trend in NCOA4 mRNA expression, significant upregulation of LC3B mRNA (P < 0.01), and a downward trend in FTH1 and P62 mRNA. Conversely, eutopic endometrial tissues showed significant downregulation in NCOA4 mRNA (P < 0.001), a downward trend in LC3B mRNA, and upregulation in FTH1 and P62 mRNA (P < 0.05 or 0.00001), as detailed in Table 4.
Table 4
Comparative mRNA Expression Levels of NCOA4, LC3B, FTH1, and P62 in Healthy, Eutopic, and Ectopic Endometrial Tissues of Rats (x¯±s)
Group
|
Number
|
NCOA4 mRNA
Relative Expression levels
|
LC3B mRNA
Relative Expression levels
|
FTH1 mRNA
Relative Expression levels
|
P62 mRNA
Relative Expression levels
|
Healthy Endometrium
|
3
|
0.94 ± 0.03
|
0.41 ± 0.06
|
1.04 ± 0.06
|
0.09 ± 0.1
|
Healthy Endometrium
|
3
|
0.28 ± 0.08***
|
0.28 ± 0.13
|
2.05 ± 0.44*
|
1 ± 0.01*****
|
Ectopic Endometrium
|
3
|
1.09 ± 0.55
|
1.01 ± 0.13**
|
1.05 ± 0.4
|
0.03
|
Note: Data are presented with two decimal places. Compared to healthy endometrium, *P < 0.05, **P < 0.01, ***P < 0.001, *****P < 0.00001. |