Austin J Reprod Med Infertil. 2021; 7(1): 1057.
Different Expressions of T-helper Cell Subsets-Related Transcription Factors and Cytokines in Infertile Women with Endometriosis
Tarokh M1, Poordast T2,3, Ghaffari-Novin M1*, are M4, Tahmasebi F4, Hesampour F4 and Gharesi-Fard B3,4,5*
1Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
4Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
5Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
*Corresponding author: Ghaffari-Novin M, Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Gharesi-Fard B, Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
Received: September 17, 2021; Accepted: November 02, 2021; Published: November 09, 2021
Purpose: It is suggested that alterations in immunologic mechanisms intervenes in the pathophysiology of endometriosis and the disease complications. There are several published reports regarding alteration in T helper cell subsets in the pathology of reproductive organs. This study was designed to find the pattern of T helper subsets in endometriosis compared with non-endometriosis women.
Methods: Expression of T helper subset-specific transcription factors (TBET, GATA3, RORC, FOXP3) and related cytokines, including IL-4, IL-10, IL-17, IL-23, TNFA, IFNG and TGFB were investigated by real time-PCR in peripheral blood, peritoneal fluid, eutopic and ectopic endometrium.
Results: The expressions of RORC, FOXP3 and all the studied cytokines were significantly increased in blood mononuclear cells of endometriosis group. The expression levels of TBET, FOXP3, IL-17A and TGFB were significantly elevated in peritoneal fluid mononuclear cells of endometriosis women. Besides, the expression levels of GATA3, RORC, FOXP3, IL-4, TNFA, and IFNG were significantly increased in eutopic endometrium of endometriosis patients. Moreover, GATA3, FOXP3 and IL-4 showed increased expressions in ectopic endometrium, while TBET, IL-23 and IFNG were significantly decreased in comparison with endometrium of non-endometriosis women. Finally, the expression levels of TBET, RORC, IL-23, TNFA, and IFNG significantly declined in ectopic endometrium in comparison with eutopic endometrium among endometriosis women.
Conclusions: Based on the present study results, it seems Th17, Th1, Th2, Treg cells and their related cytokines may play influencing role in progression of endometriosis and its symptoms including infertility. Indeed, cytokine network imbalance may cause the disease progression in patients during several years.
Keywords: Endometriosis; Infertility; T cell subsets; Transcriptional Factors
PB: Peripheral Blood; PF: Peritoneal Fluid; Th: T helper; Treg: T regulatory; IL: Interleukine; IFN: Interferone; TGF: Transforming Growth Factor; TF: Transcriptional Factor; TNF: Tumor Necrotizing Factor; PBMC: Peripheral Blood Mononuclear Cell; PFMC: Peritoneal Fluid Mononuclear Cell; Eeu: eutopic Endometrium; Eec: ectopic Endometrium; FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; PRL: Prolactin; TSH: Thyroid Stimulating Hormone
Endometriosis is a common chronic inflammatory pelvic disease in women at reproductive age with two main manifestations, including pain and infertility [1,2]. This disease is defined as the presence of endometrial tissue out of the uterine cavity . Although the exact etiology of endometriosis is not yet well known, the most accepted theory is retrograde menstruation, which could initiate immune system responses to ectopically implanted endometrial cells on the peritoneal lining among 10 to 15% of women [3,4]. It is suggested that alterations in immunologic mechanisms intervenes in the pathophysiology of endometriosis and the disease complications . In women with endometriosis, significant changes have been reported in population of immune cell subsets and types of responses in the Peripheral Blood (PB), Peritoneal Fluid (PF) as well as endometrial tissues [5,6]. Various leukocytes reside in eutopic endometrium and also endometriotic implants, and produce different cytokines and other immune mediators [5,7]. There are several reports regarding deviation of Th cells responses in endometriosis [8- 10]. While predominance of Th1 cells may promote inflammation, increase in Th2 cells modulates inflammation in affected areas. Th1 cells potentiate inflammation by producing IFN-γ. Depending on the level of secretion, IFN-γ and TNF-a are proposed to have dual effects on early stage of gestation and implantation . On the other hand TNF-a has toxicity on sperm and egg cells and activates Th1cells . TNF-a may also be produced by Th1. Th2 cells secrete IL-4, IL-10, and TGF-β as anti-inflammatory cytokines. Increase of IL-4 and IL- 10 is reported in some research on endometriosis . In addition, Th17 and regulatory T (Treg) cell subsets have been also proposed to play roles in pathogenesis of endometriosis [13-15]. Th17 cells may trigger inflammation by production of neutrophil attractive chemokines . It is argued that Th17 cells deal with induction of many autoimmune diseases, including arthritis, inflammatory bowel disease, psoriasis, uveitis, and multiple sclerosis [17,18]. IL-17A is the most important cytokine produced by Th17 cells, which mediates neutrophilic inflammation .
Increase in Treg cells inhibits the recruitment of effector immune cells. Treg cells also produce anti-inflammatory IL-10 and TGF-β cytokines . It is reported that women with endometriosis show decreased level of cytokines IL-17 and TGF-β some weeks after laparoscopy . This is also followed by increase in pregnancy rate during next months.
Moreover, it is well documented that Th cells and their related cytokines cooperate with each other as a network. So, disturbance in Th subsets could be accounted as a major immunological etiology for many infertility-related diseases including endometriosis [21,22]. In line with this idea, several studies have shown that the proportion of immune cells and cytokine production in women with endometriosis has altered in comparison with healthy women [23,24]. In addition to signature cytokines, investigation of Th subset-specific Transcription Factors (TFs) has been also conducted with the aim of evaluating the Th balance in endometriosis [25,26].
Interestingly, there is no published data regarding the simultaneous investigation and comparison of the Th subsets balance in all the potential samples, including PB, PF, intra (eutopic), and extra (ectopic) uterine endometrial tissue samples in endometriosis patients. In the previous published research, only one or two source of PB, PF, eutopic or ectopic uterine endometrial tissues have been investigated. So, in the present study, the expression of TFs and cytokines related to Th1, Th2, Th17, and Treg in PB, PF, and endometrial tissues were simultaneously investigated to elucidate the pattern and the role of T cell subsets involved in endometriosis pathogenesis. For this purpose, we analyzed and compared genes expression of TBET, GATA3, RORC, FOXP3, IL-4, IL-10, IL-17, IL- 23, TNFA, IFNG, and TGFB from endometriosis and healthy women in all the mentioned samples.
This study was approved by the Medical Ethics Committees of both Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.SM.REC.1394.79) and Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1395.S307). All participants read and signed written informed consent before entering this research study and sampling. Clinical and demographic features of the participants are shown in Table 1.
Cause of laparoscopy
Tuba ligation, hemorrhage, Myoma
Known autoimmune diseases
PB, PF, Eeu, Eec
PB, PF, Eeu
E: Endometriosis; NE: Non-Endometriosis; PB: Peripheral Blood; PF: Peritoneal Fluid; Eeu: Eutopic Endometrium; Eec: Ectopic Endometrium.
Table 1: Clinical and demographic features of women participated in this study.
In this study, two groups of women were compared. Both groups were at reproductive age (20-45 years old) and had not received either any anti-metabolism and immune-suppressive medications or hormonal therapy three months prior to laparoscopy. Based on the medical records, the subjects had no history of autoimmune diseases or malignancy. Group I included 30 infertile women with late-stage endometriosis.
Infertile late endometriosis group consisted of patients with stages III and IV according to the revised American Society for Reproductive Medicine (rASRM) classification. Confirmation and staging of endometriosis were determined through laparoscopy by the same surgical team including two expert gynecologic laparoscopists. The patients have had usual unprotected intercourse for at least one year without pregnancy and had no other causes for infertility on the basis of spermogram, pelvic sonography, and hormonal assays (FSH, LH, TSH, and PRL). In this study group, peripheral blood, peritoneal fluid, eutopic (Eeu), and ectopic (Eec) endometrial tissue samples were obtained. Group II included 30 fertile women as the fertile non-endometriosis. This control group consisted of women, who had at least two children and underwent laparoscopy due to legal tubal ligation, myomectomy, or nonstop bleeding hysterectomy. In this group, no endometriotic foci or immunologic pathology were observed during laparoscopy. The obtained samples from fertile nonendometriosis group were similar to endometriosis group except lack of ectopic endometrium.
Women who had the described criteria of one of the study group in their records and were taken to operating room for laparoscopy were put in the appropriate list. Before general anesthesia, 10mL of peripheral venous blood was aspirated by a heparinized syringe and transferred into a sterile tube. After induction of anesthesia and insertion of trocars and before any other manipulation, 2 to 10 mL of peritoneal fluid was aspirated from Douglass pouch and treated with heparin. All the bloody samples were excluded from the study. Mononuclear cells of both PB (PBMCs) and PF (PFMCs) were collected using Ficoll hypaque and density gradient centrifugation at 3000 rpm for 20 minutes. The aliquots containing two million cells were stored at -70°C until RNA extraction. Moreover, extra-uterine endometrial tissue samples of pelvic endometrium were obtained from endometriosis patients. One part of these tissue samples was laid in formalin for the ultimate histopathological confirmation of the disease, and the other part was placed in physiological serum for RNA extraction. Finally, eutopic endometrial tissue samples were collected by curettage from both groups. All these tissue samples were washed adequately to remove blood and stored at -70°C until RNA extraction.
To reach an accurate result in real time-PCR, items of MIQE guidelines were considered. Total RNA from PBMCs, PFMCs, eutopic, and ectopic endometrial tissue samples were extracted by using a commercial Total RNA extraction kit (Parstous, Mashhad, Iran) according to the manufacturer instruction. Possible genomic DNA was removed by treating RNA samples with RNase-free DNase I (Thermo Fisher Scientific, Carlsbad, USA). cDNA was also synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, Carlsbad, USA). Using Genevestigator biomedical software, RPL13a and ACTB were determined as reference genes for mononuclear cells and endometrial tissue samples, respectively. Primer sequences were selected by means of Allel ID 7.5 software and primer-BLAST (Table 2). Furthermore, real time-PCR amplifications were performed using Applied Biosystems SYBR Green (Thermo Fisher Scientific, Carlsbad, USA) and ABI 7500 real time-PCR system (Applied Biosystems, Foster City, California, USA). Real time-PCR amplification programs were as follows: 95°C (10') 94°C (30'') 60°C (1') for TBET, GATA3, FOXP3, TNFA, TGFB, IL-4 and 95°C (10') 95°C (15'') 60°C (30'') 72°C (30'') for IL-10, IL-17, IL-23, RORC, IFNG, RPL13a, ACTB.
Table 2: Sequences of primers used for qRT-PCR.
Statistical analyses were performed using IBM SPSS software Version 18. Normality of the data was assessed by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Probability values were calculated on the basis of two-tailed tests. All differences in fold changes were analyzed by the non-parametric Kruskal-Wallis test (pot-hoc: Mann- Whitney U-test). The correlations between the expression levels of different studied genes were calculated by Pearson’s correlation test.
Fold Change, ΔΔCt, and ΔCt were calculated by the following formula in which E and NE are representatives of endometriosis and non-endometriosis groups respectively.
ΔCtE = CtE (target gene) - CtE (reference gene)
ΔCtNE = CtNE (target gene) - CtNE (reference gene)
ΔΔCt = ΔCtE - ΔCtNE
Fold Change = 2-ΔΔCt
Expression of TFs in PBMCs and PFMCs: Figure 1 and 2 indicate the results of expression of Th cells’ TFs between endometriosis and non-endometriosis groups using PBMCs and PFMC samples. As shown in Figure 1, the expression levels of RORC and FOXP3 in PBMC samples were significantly increased in endometriosis group compared with the non-endometriosis group (P=0.0001 for both TFs).
Figure 1: Genes expression of transcription factors in PBMCs of women with endometriosis (E) in comparison with non-endometriosis women (NE).
Regarding PFMCs samples in Figure 2, TBET and FOXP3 have significantly over-expressed in endometriosis group (P=0.001 and P=0.0001, respectively).
Figure 2: Genes expression of transcription factors in PFMCs of women with endometriosis (E) in comparison with non-endometriosis women (NE).
We, additionally, analyzed the ratios between the expression of the studied inflammatory and anti-inflammatory transcription factors in both PBMC and PFMC samples (Table 3). Within PBMC samples, the ratios of TBET Δct/GATA3 Δct, and TBET Δct/FOXP3 Δct were significantly higher in endometriosis in comparison with non-endometriosis group (P=0.004 and P=0.0001, respectively). Regarding PFMC samples, TBET Δct/GATA3 Δct ratio was significantly decreased in endometriosis group (P=0.0001). However, the ratio between sum of inflammatory and sum of antiinflammatory TFs (Δct TBET + Δct RORC/Δct GATA3 + Δct FOXP3) was significantly lower in endometriosis women in both PBMC and PFMC samples (P=0.0001 for both comparisons).
Ratios of TF Δct
TBET Δct/GATA3 Δct
TBET Δct/FOXP3 Δct
TBET Δct +RORC Δct/GATA3 Δct + FOXP3 Δct
TF: Transcriptional Factor; NE: Non-Endometriosis Women; E: Endometriosis.
Table 3: The ratios of inflammatory to anti-inflammatory TFs in PBMC and PFMC of endometriosis and non-endometriosis groups.
Also, in terms of PBMCs in the control group, there were significant positive correlations between Δct of TBET and two TFs GATA3 and FOXP3 (P=0.0001 and P=0.003 respectively), between GATA3 and FOXP3 (P=0.001), and between FOXP3 and RORC (P=0.0001, Table 1 in the supplemental file). All the TFs were also significantly correlated with each other in PFMC of this group (P=0.0001 for all the correlations, Table 1 in the supplemental file). On the other hand, in PBMC of endometriosis patients, there was only significant positive correlation between GATA3 and FOXP3 (P=0.012). In PFMCs of this group, however, all the TFs were significantly correlated with each other (P=0.0001 except between TBET and RORC with P=0.006, Table 2 in the supplemental file).
Expression of TFs in endometrial tissues: Results of the expression of TFs in endometrial tissues are presented in Figure 3. As indicated, the expression of GATA3, RORC, and FOXP3 were significantly increased 2, 1.08, and 2.7 folds, respectively, in eutopic endometrial tissue from endometriosis patients compared with non-endometriosis control group (P=0.007, P=0.017, P=0.0001). Regarding ectopic tissues, the expression of GATA3 and FOXP3 were increased 2.38 and 2.76 folds, respectively (P=0.015, P=0.0001), while TBET was significantly down regulated (P=0.0001) in women with endometriosis compared with non-endometriosis control group.
Figure 3: Genes expression of transcription factors in eutopic and ectopic endometrial tissues (Eeu and Eec) of women with endometriosis in comparison with endometrium and non-endometriosis women (NE).
When comparing ectopic and eutopic tissues, the expression of inflammatory TFs TBET and RORC significantly decreased in ectopic tissue of endometriosis patients (P=0.0001, P=0.037).
Analysis of the ratios of inflammatory to anti-inflammatory TFs and comparison of these ratios in endometrial tissues are presented in Table 4. TBET Δct / GATA3 Δct was significantly higher in ectopic tissue than endometrium of control and endometriosis groups, almost 2.2 and 19.6 times respectively (P=0.0001 for both comparisons). Moreover, TBET Δct / FOXP3 Δct significantly increased by over 97 times in eutopic endometrium of endometriosis patients compared with the control group (P=0.003). Also, TBET Δct / FOXP3 Δct in ectopic endometrial tissue of endometriosis women significantly rose 15 times compared with endometrium of the control group (P=0.0001), it significantly declined in comparison with eutopic endometrium of the same group, though (P=0.046). Similarly, the ratio between sum of inflammatory TFs expression and the sum of anti-inflammatory TFs was significantly increased in eutopic endometrium of endometriosis women versus endometrium of the control group (P=0.001). This ratio indicated decreasing in ectopic endometrial tissues of endometriosis patients in comparison with endometrium of the same group (P = 0.013).
Ratios of fold changes of Endometrial tissues TFs
Mean of Ratios of 2-ΔΔct (±SEM)
NE vs. Eeu
NE vs. Eec
Eeu vs. Eec
TBET Δct/GATA3 Δct
TBET Δct/FOXP3 Δct
TBET Δct + RORC Δct/GATA3 Δct + FOXP3 Δct
TFs: Transcriptional Factors; NE: Non-Endometriosis Women; E: Endometriosis.
Table 4: Ratios of inflammatory to anti-inflammatory TFs Δct and fold change between different endometrial tissues.
Likewise, significant positive correlations were found between Δct of (GATA3 and TBET), (GATA3 and FOXP3), and (GATA3 and RORC) in endometrium of the control group (P=0.006, P=0.0001, P=0.015, respectively). In eutopic endometrium of endometriosis patients, correlations between (FOXP3 and GATA3) as well as (FOXP3 and RORC) were significantly positive (P=0.034, P=0.015). In ectopic endometrium of endometriosis patients, only GATA3 and RORC displayed a significant positive correlation (P=0.001, Table 3 in the supplemental file).
Cytokines in PBMC and PFMC: Concerning PBMC, Figure 4 shows that the expression of cytokines IL-17, IL-23, TNFA, and TGFB significantly increased (P=0.001 for IL-17 and P=0.0001 for the others) in endometriosis patients, while expressions of IL-10 and IFNG significantly decreased to 0.95 and 0.85, respectively (P=0.048, P=0.032). On the other hand, considering PFMC in Figure 5, the expression of IL-17 and TGFB significantly increased 2 and 8 times in endometriosis women, respectively (P=0.025, P=0.0001).
Figure 4: Genes expression of cytokines in PBMC of women with endometriosis (E) in comparison with non-endometriosis women (NE).
Figure 5: Genes expression of cytokines in PFMC of women with endometriosis (E) in comparison with non-endometriosis women (NE).
Despite using different IL-4 primer sets, the product of IL-4 gene was not detected by qRT-PCR in PBMC and PFMC samples.
Besides, in the control group, there were significant positive correlations between Δct of all cytokines in PBMC (different P values) with two exceptions between IL-17 and TNFA as well as IL-17 and IFNG. Considering PFMCs of control women, all cytokines were positively correlated (P=0.0001, Table 4 in the supplemental file).
On the other hand, in PBMC of endometriosis women, there were significant positive correlations between cytokines except for TNFA. Also, there was no correlation between IL-10 and IFNG and between TGFB and IFNG. In PFMC of this group, significant positive correlations were observed between all cytokines (different P values) except TNFA and IL-17 as well as between TNFA and IFNG (Table 5 in the supplemental file).
Cytokines in endometrial tissues: Results of the investigation of cytokines in endometrial tissues are presented in Figure 6. Expression of IL-4 increased in eutopic and ectopic tissues of endometriosis patients compared with endometrium of the control group (P=0.012, P=0.010). Expression of IL-23 decreased in ectopic endometrial tissue of endometriosis women compared to eutopic endometrium of both endometriosis and non-endometriosis women (P=0.007, P=0.0001, respectively). Regarding TNFA, it increased in eutopic endometrium of patients compared with non-endometriosis tissue, but it decreased in ectopic endometrial tissue compared with eutopic of endometriosis patients (P=0.0001 for both comparison). Compared with endometrium of the control group, expression of IFNG increased 3 times in eutopic endometrium of endometriosis patients (P=0.003). On the other hand, this cytokine decreased in ectopic endometrial tissue compared to eutopic endometrium of the same group and also non-endometriosis women (P=0.0001, for both).
Figure 6: Genes expression of cytokines in eutopic (Eeu) and ectopic (Eec) endometrial tissues of women with endometriosis in comparison with endometrium and non-endometriosis women (NE).
In endometrium of control group, Δct of IL-4 and IFNG had significant positive correlation (P=0.006). There was also significant positive correlation between IL-10 and TGFB (P=0.020).
Regarding eutopic endometrium of endometriosis patients, IL-4 and TNFA had significant positive correlations (P=0.014). Finally, in ectopic endometrial tissue of the patients, IL-4 and IL-17 were significantly correlated (P=0.047). In addition, IL-17 had significant positive correlations with IL-10, IL-23, and TNFA (P=0.029, P=0.011, P=0.025). Also, we noticed significant positive correlations between IL-23 and IL-10 as well as between IL-23 and TNFA (P=0.007, P=0.010, Table 6 in the supplemental file).
Contrarily, there were significant negative correlations between Δct of IL-10 in eutopic endometrium and IL-4, IL-10, and TGFB in ectopic endometrial tissue in endometriosis patients (P=0.022, P=0.042, P=0.019 respectively). Also, a significant negative correlation was found between IFNG in eutopic and IL-23 in ectopic endometrial tissues in endometriosis patients (P=0.034, Table 7 in the supplemental file).
Endometriosis is a hormone-dependent disease in which immune cell populations change especially in four major parts: PB, PF, endometrium, and peritoneal implants. Subpopulations of T cells that reside in these parts determine immune responses and the disease manifestations, such as infertility and pain . Imbalance between Th1 and Th2 cells, and decrease of Treg cells have been suggested for establishment of endometriosis .
Considering PBMCs in this study, we found that the elevations of RORC and FOXP3 were significant. Previously, in accord with our findings, Ahn SH et al. and Hirata T et al. reported increased level of IL-17, the main cytokine of Th17, in the serum of endometriosis patients [20,29]. These findings may highlight the role of Th17 cells in the pathogenesis of endometriosis. However, regarding FOXP3, our finding was not consistent with what Olkowska-Truchanowicz et al. found in their study probably because they selected endometrioma patients . Besides, there is no consensus about the frequency of Treg cells or increase of FOXP3 expression in PB .
Regarding PFMCs, in the present study, increases in mRNA expressions of TBET and FOXP3 were significant. In this regard, Ho et al. announced that all subsets had increased in endometriosis group compared with the control group, but Th1 cells had increased less than other subsets . Richter ON et al. and Othman EER et al. also reported increase in Th1 axis cytokines, including TNF-a and IFN-γ [32,33]. As peritoneum is the main area of inflammation, increase of Th1 cells in PF is expectable. Also, the increased level of FOXP3 in the PF was in accordance with the result of other studies that showed elevated frequency of Treg cells in the PF [15,34]. Some previous research had conflicting results regarding Treg cells of PB and PF between endometriosis and control groups . Logically, as endometrial implanted cell, antigens are in close contact with the active immune cells in the peritoneal lining and fluid, these cells should be more responsive than those in the blood circulation. Therefore, increased frequency of Treg cells might be a regulatory and inhibitory mechanism in pelvic peritoneum.
Furthermore, regarding the correlations between TFs of T cell subsets in PBMCs and PFMCs, a network mechanism was observed. Significant correlations between TFs of PBMCs in healthy women showed that a potent regulated mechanism exists between TFs, but in endometriosis women, only the correlation between GATA3 and FOXP3 was significant. This could be explained as the disruption of the cytokine and TFs network mechanism in the patients. Considering PFMC, all the TFs were significantly correlated in both study groups. This might also be the result of close cooperation of different immune cells in the peritoneum.
The results of the present study regarding gene expressions and correlations in PBMCs and PFMCs indicated that both inflammatory and anti-inflammatory subsets are activated. In addition, the similarity of diagrams shows the presence of a systemic immune response even though presentation of inflammation in endometriosis is mainly focused in pelvic cavity.
In this study, comparison of eutopic endometrium of endometriosis patients with the control group revealed significant increases in mRNA expressions of GATA3, FOXP3, and RORC. In accordance with the current study, Podgaec S. et al. and Chen P. et al. found increased expression of GATA3 and Th2 cytokines in endometriosis [10,36]. So, endometriosis can be considered as an inflammatory disease with Th2 dominance . In addition, in the present study, the increased level of FOXP3 T cells in endometrial tissues of endometriosis patients is in accordance with findings of Berbic M et al. . However, previous studies presented conflicting results on FOXP3 gene expression or Treg cell frequency in eutopic endometrium [28,38]. For instance, Jasper et al. reported a decrease of FOXP3 expression in the secretory phase , which is not in consistence with our result, as we investigated FOXP3 expression in proliferative phase. Also, we found that the expression of RORC was higher in endometrium of endometriosis patients in comparison with endometrium of non-endometriosis group. This is in line with the Takamura’s study and shows that Th17 cells have a major role in endometriosis . In line with most of the previous studies, we did not find significant increase of TBET expression in eutopic endometrium of endometriosis patients. Inman D et al. showed cyclic presentation of TBET and GATA3 in normal endometrium during menstrual cycle; hence, time of sampling as well as causes of infertility in control group can influence on the results . For example, Koval H et al. found an increase in TBET and GATA3, but they reported decrease in FOXP3 expression. In this research, they compared endometriosis patient with infertile women with tubal causes . Also, many other studies recognized increases in Th1- related cytokines.
Considering ectopic endometrial tissue, we noticed that the expression of GATA3 and FOXP3 significantly increased in comparison with endometrium of the control group, but TBET decreased significantly. Takamura et al. also reported decrease of Th1 cells in ectopic endometrial tissue . As the expressions of TBET and GATA3 are regulated in the opposite manner, increase of GATA3 and decrease of TBET are expected observations [41,42]. In line with current study results, Podgaec S et al. and Chen P et al. reported increase of GATA3, Th2 cells and their related cytokines in ectopic lesions [26,43]. Regarding FOXP3, Budiu et al. reported increased expression of this molecule in the context of endometriosis . Besides, two other experiments reported an increase in the frequency of Treg cells in ectopic endometrial tissues [37,45].
In this study, TBET and RORC decreased significantly in ectopic endometrial tissue in comparison with eutopic tissue in endometriosis patients. We did not find any papers on TBET gene expression in ectopic endometrial tissues, but Takamara et al. found that the proportion of Th1 cells in ectopic lesions had been significantly lower than eutopic endometrium . As to RORC, our finding did not support that of Takamura et al. since they found higher Th17 cells in ectopic lesion in comparison with endometrial tissue . Frequency of these cells was also higher in PF of endometriosis patients and in association with the severity of the disease . Then according to the current study, increase of Th17 cells is prominent in PF and not within the lesions. Significant decrease of inflammatory TFs in ectopic lesions seen in this study might be due to suppression by active anti-inflammatory factors.
Some of the previous studies found that the ratio of Th1/Th2 could be more informative than frequency of a subset or expression of a single TF to discriminate the disease [42,47]. Meanwhile, some research reported the imbalance between Th1/Th2 cells in endometriosis. For example, Siedentopf et al. noticed a higher Th1/Th2 ratio in the PB of patients affected by endometriosis . Regarding the ratios of inflammatory to anti-inflammatory TFs (subsets), this study showed that TBET/GATA3 and TBET/FOXP3 ratios were higher in PBMCs of endometriosis patients in comparison with the control group. This certified that inflammation had occurred systemically in endometriosis patients. On the other hand, in PFMCs of these patients, all of the inflammatory to anti-inflammatory ratios decreased in comparison with the control group. These ratios suggested that severity of inflammation has especially provoked activation of braking cells and more potent anti-inflammatory pathways in PF.
In eutopic endometrium of endometriosis patients in comparison with endometrium of the control group, TBET/FOXP3 as well as the TBET + RORC/GATA3 + FOXP3 ratios significantly decreased. These ratios might suggest that regulatory pathways, especially Treg cells are more active in endometriosis patients. Comparing ectopic endometrial tissue with endometrium of the control group, TBET/ GATA3 and TBET/FOXP3 ratios significantly increased. On the other hand, comparison of ectopic and eutopic endometrial tissues revealed that TBET/GATA3 ratio significantly rose in ectopic tissue, but TBET/ FOXP3 and TBET+RORC/GATA3+FOXP3 ratios decreased. These findings might also propose that inflammation is augmented in extrauterine lesions but potently suppressed by anti-inflammatory factors.
In the present study, moreover, all the investigated cytokines expressed differently in PBMCs of endometriosis patients compared with non-endometriosis women. The mRNA expressions of IL-10 and IFNG decreased significantly in endometriosis patients, while mRNA expression of other cytokines increased. The findings about IL-10 mRNA expression is not in accordance with the study by Antsiferova et al. , but it is in line with findings of two other experiments that reported decreased IL-10 level in late-stage of endometriosis [50,51]. Likewise, with regard to IFN-γ, Othman et al. reported an increase in this cytokine in PB of endometriosis patients , but most of others did not report any difference or decrease of this cytokine . On the other side, in accordance to our results increase in TNF-a serum was reported in many studies. Some of them showed association between TNF-a level and severity of endometriosis [43,53]. The elevated expression of TGFB was also in line with Kyama CM’s investigation . TGF-β is a cytokine produced by Treg cells and necessary for the differentiation of these cells, and this reciprocal interaction can validate the associated increase of TGFB and FOXP3 in this study. According to reports, the increased level of IL-23/IL-17 pathway is an important axis of inflammation and increase of IL-17 should be considered as an initiation factor for the disease . We found that this cytokine increased significantly in the PBMCs of endometriosis patients, but it is not in line with Andreoli’s study, probably because they selected patients with the early-stage of the disease .
With respect to PFMCs, we found that the cytokines expression had a similar pattern with PBMCs. This similarity may accentuate endometriosis has a systemic response associated with concentration in peritoneum. However, the increase in the expression level of IL- 17 and TGFB was significant. These findings are in agreement with those of Zhang et al. and Kyama et al. [54,57]. According to research, these two cytokines have crucial roles in some diseases with infertility consequences .
On the other hand, there were significant increases in IL-4, TNFA and IFNG mRNA expression in eutopic endometrium of patients in comparison with the control group. Antsiferova et al. indicated diminished mRNA expression of IL-4 in eutopic endometrium of endometriosis patients. This is in disagreement with our results, although they did not mention staging of the patients . Kyama et al. also found an increase in TNF-a , but there is a conflicting study that reported decrease of TNF-a level in endometriosis patients compared with healthy controls . Our results concerning IFN-γ in eutopic endometrium provide more evidence for the other studies . Increased TNF-a plays role in infertility and IFN-γ does likewise.
Comparing ectopic endometrium of patients with the endometrium of non-endometriosis women, we found that IL-4 increased significantly, while IL-23 and IFNG decreased dramatically. OuYang et al. also reported abundance of IL-4 positive cells in stroma of ectopic endometrium in endometriosis patients . Other researchers also indicated that IL-4 and TGF-β1 increased in endometriotic lesions [49,62,63]. So, decreased level of inflammatory cytokines in extra-uterine lesions might indicate an overwhelming anti-inflammatory condition that facilitates establishment and expansion of the lesions. In agreement with our study, Mier-Cabrera et al. reported that increase of IFN-γ in mouse models could reduce weight and area of ectopic lesions . Also, Szyllo et al. showed diminished secretion of IFN-γ from stimulated lymphocytes of endometriosis patients in comparison with control group. No article was found about expression of IL-23 in ectopic lesions of endometriosis.
On the other hand, gene expression of IFNG significantly decreased in ectopic endometrium of endometriosis patients in comparison to eutopic endometrium of the same group. However, the result is in disagreement with that of Chiang et al., in that, they found enhanced IFN-γ expressing T cells in ectopic lesions compared to eutopic endometrium . But Bergqvist’s et al. finding was in accordance with our study . This reduction, as well as decrease of IL-23 and TNFA, may be the result of increased level of TGF-β, as this cytokine inhibits inflammation through suppressing Th1 and Th17 [63,66].
In summary, this work’s findings along with the previous studies suggest that at first endometrial cells enter peritoneal cavity during retrograde menstruation. As displaced antigens, endometrial cells stimulate native immunity to send signals by producing cytokines. Then, by activating Th17 cells, neutrophilic inflammation initiates, which is intensified by Th1 cells. At this stage, inflammatory cytokines hurt the surrounding tissues. As a result of cyclic hormonal changes that lead to GATA3 expression, activated Th2 cells produce antiinflammatory cytokines to decelerate the inflammation. At last, gene expression of FOXP3 as well as production of TGF-β activates Treg cells to subside inflammation and repair damaged tissues through fibrosis. After several years of repetitive increase and decrease of inflammation in every menstrual cycle, fibrosis and organ attachment take place. Indeed, no specific T cell subset alone could be responsible for the establishment and progress of endometriosis, but periodically all the four subsets engage in the process of endometriosis in turn and in a network manner. Disruption of this network mechanism could contribute to the progress of the disease stages in patients.
Comparison between endometriosis and non-endometriosis women shows Th17, Th1, Th2 and Treg cells through IL-17, TNF-a, IL-4, and TGF-β have influencing effect on the disease progress. While inflammatory T helper cells and cytokines are actively impressive in initiation and progression of endometriosis, suppressing T helper cells and cytokines may inhibit inflammation especially in endometriotic tissues.
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Citation: Tarokh M, Poordast T, Ghaffari-Novin M, Zare M, Tahmasebi F, Hesampour F, et al. Different Expressions of T-helper Cell Subsets-Related Transcription Factors and Cytokines in Infertile Women with Endometriosis. Austin J Reprod Med Infertil. 2021; 7(1): 1057.