Skip to main content

Altered gene expression of VEGF, IGFs and H19 lncRNA and epigenetic profile of H19-DMR region in endometrial tissues of women with endometriosis

Abstract

Background

Endometriosis, as chronic estrogen-dependent disease, is defined by the presence of endometrial-like tissue outside the uterus. Proliferation of endometrial tissue and neoangiogenesis are critical factors in development of endometriosis. Hence, vascular endothelial growth factor (VEGF) as well as insulin‐like growth factor 1 and 2 (IGF1, 2) may be involved as inducers of cellular proliferation or neoangiogenesis. Imprinted long noncoding RNA H19 (lncRNA H19) has been suggested to be involved in pathogenesis of endometriosis via regulation of cellular proliferation and differentiation. Epigenetic aberrations appear to play an important role in its pathogenesis. The present study was designed to elucidate VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of differentially methylated region (DMR) of H19 (H19-DMR) regulatory region in endometrial tissues of patients with endometriosis, in comparison with control women.

Methods

In this case–control study, 24 women with and without endometriosis were studied for the relative expression of VEGF, IGF1, IGF2 and H19 lncRNA genes using real-time polymerase chain reaction (PCR) technique. Occupancy of the MeCP2 on DMR region of H19 gene was assessed using chromatin immunoprecipitation (ChIP), followed by real-time PCR.

Results

Genes expression profile of H19, IGF1 and IGF2 was decreased in eutopic and ectopic endometrial tissues of endometriosis group, compared to the control tissues. Decreased expression of H19 in ectopic samples was significant in comparison with the controls (P < 0.05). Gene expression of VEGF was increased in eutopic tissues of endometriosis group, compared to control group. Whereas its expression level was lower in ectopic lesions versus eutopic and control endometrial samples. ChIP analysis revealed significant and nearly significant hypomethylation of H19-DMR region II in eutopic and ectopic samples, compared to the control group respectively. This epigenetic change was aligned with expression of IGF2. While methylation of H19-DMR region I was not significantly different between the eutopic, ectopic and control endometrial samples.

Conclusion

These data showed that VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of H19 lncRNA have dynamic role in the pathogenesis of endometriosis, specifically in the way that hypomethylation of H19-DMR region II can be involved in IGF2 dysregulation in endometriosis.

Peer Review reports

Plain language summary

Endometriosis as an estrogen-dependent chronic inflammatory disease is characterized by the growth of endometrial-like tissue outside the uterus. In this study, to evaluate the effect of genetic and epigenetic factors involved in this disease, VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of H19-DMR regulatory region in endometrial tissues of 12 patients with endometriosis and 12 normal women (as control) were assessed. The results showed that expression of H19, IGF1 and IGF2 genes was decreased in eutopic and ectopic endometrial tissues of endometriosis group in comparison with control group. Expression of VEGF gene was increased in eutopic tissues of endometriosis group compared to the control group. Whereas its expression level was lower in ectopic lesions versus eutopic and control endometrial samples. Methylation of H19-DMR region II was decreased in eutopic and ectopic samples compared to the control group. This epigenetic change was aligned with IGF2 expression. Methylation of H19-DMR region I was not significantly different between eutopic, ectopic and control endometrial samples. Findings of this study showed that VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of H19 lncRNA have dynamic role in the pathogenesis of endometriosis. Additionally, hypomethylation of H19-DMR region II may be involved in impaired IGF2 regulation in endometriosis.

Background

Endometriosis is an estrogen-dependent gynecological disease in women of child-bearing age. This chronic disease is defined as presence of endometrial glands and stromal cells outside the uterine cavity [1]. Endometriosis affects 6–10% of women in reproductive-age. Endometriotic lesions are often found in the ovaries, fallopian tubes and peritoneal cavity [2]. Pelvic pain and infertility are common symptoms of endometriosis. Other symptoms of endometriosis include dysmenorrhea, irregular menstruation, dyspareunia and dysuria [3]. Endometriosis is considered as a multifactorial disease affected by genetic, hormonal, immunological and environmental factors [4]. On the basis of previous studies, adhesion, proliferation of endometrial cells, cellular invasion and neoangiogenesis are key factors in the pathogenesis of endometriosis [5]. Therefore, growth factors including insulin-like growth factors (IGFs) may play roles in inducing of cellular proliferation and differentiation [6]. Endometrial tissue produces IGF1 and IGF2, playing important roles in growth and differentiation of endometrial cells [7]. It has been shown that IGF1 prevents apoptosis and acts as a mitogenic factor for endometrial cells [8]. In addition, it has been reported that IGF-1 protein concentration is increased in the peritoneal fluid of patients with endometriosis compared to control women [9]. IGF2 gene is imprinted and paternally expressed in prenatal mammalian tissues [10]. It has been shown that IGF1 deficiency causes infertility and hypoplasia of uterus in female mice, and it was suggested that IGF1 has main role in uterine growth and function [6]. Human endometrial epithelial and stromal cells express IGF1 and IGF2 that their highest expression levels are in the late and early proliferative phase, respectively [11]. It has been suggested that angiogenesis is necessary for growth and survival of endometriotic lesions [12]. Vascular endothelial growth factor (VEGF) is the most important angiogenesis factor that causes endothelial proliferation, vasodilation and increases vascular permeability [13]. Several researches showed that level of VEGF mRNA is higher than control group, in endometriosis, which indicated the main role of VEGF in angiogenesis related to endometriosis [14]. Long noncoding RNAs (lncRNAs) are non-protein-coding transcripts with longer than 200 nucleotides. This type of RNAs are involved in important functions of various biological processes in cancer [15]. H19, as lncRNA, is located in an imprinting region on chromosome 11p15.5 of human. It plays a major role in embryonic development and regulating growth. H19 is expressed from maternally allele [16]. Structurally, H19 gene includes five exons and four introns that produce a 2.3-kb lncRNA after splicing [17]. In the human endometrium, expression of H19 increases at the late proliferative phase [18]. In recent years, studies suggested that endometriosis can be considered as an epigenetic disease which involves DNA modifications. Epigenetics is composed of heritable phenotype changes which are not caused by alterations in the DNA sequence. Evidences suggested that various epigenetic modifications, such as DNA methylation, may play a main role in the etiology of endometriosis [19]. DNA methylation generally acts to suppress gene transcription which occurs through binding methylated DNA-binding proteins, such as methyl CpG binding protein 2 (MeCP2), to methyl-CpG sites [20]. H19 and IGF2 are reciprocally imprinted genes. IGF2 gene is located 90 kb away from H19 gene. Expression of these two genes is coordinately regulated through an intergenic differentially methylated region (DMR) and downstream enhancers. DMR region, also called imprinting control region (ICR), are located in 2–4 kb upstream of the H19 transcription site. So that, H19 is expressed from maternal allele while IGF2 is expressed from the paternal allele [21].The responsible mechanism for controlling H19 and IGF2 imprinting consists of binding MeCP2 or CCCTC-binding factor (CTCF) based on the methylation status of DMR. This region includes seven binding sites for insulating factor CTCF, which methylation changes in the sixth CTCF-binding site is related to the altered expression of H19 and IGF2 genes in diseases [22]. On the paternal chromosome, binding MeCP2 to the hypermethylated DMR causes gene expression of IGF2 and repression of H19. Conversely, on the maternal chromosome, H19 is expressed and IGF2 is repressed by CTCF protein binding to the hypomethylated DMR [23]. H19 regulates gene expression through epigenetic mechanisms by interacting with chromatin-modifying complexes. Therefore, lncRNA H19 could play role in the pathogenesis of some diseases [24]. The role of H19 was showed in infertility related diseases. Korucuoglu et al. showed that H19 expression is reduced in endometrial tissues of unexplained infertile women [25], while Ghazal et al. revealed that H19 regulated endometrial tissue proliferation by altering IGF signaling in endometriosis [26]. In the present study, we evaluated and compared expression levels of H19 RNA factor and angiogenic (VEGF) and proliferative (IGF1, IGF2) genes in endometrium of non-endometriosis women in comparison with eutopic and ectopic tissues of endometriosis women. Additionally, we investigated modifications of DNA methylation (MeCP2 incorporation) of the H19-DMR regulatory region in these tissues.

Methods

Subjects and tissue samples

In this case control study, 24 women undergoing diagnostic laparoscopy at Royan Institute, (Tehran, Iran) were enrolled from 2019 to 2020. According to diagnostic laparoscopy findings, 12 women with endometriosis and 12 women without endometriosis were respectively considered as endometriosis and control groups. All women were 20–45 years old and they had no endometrial hyperplasia or neoplasia. In addition, none of them did receive hormonal drugs within the last three months. Women without endometriosis in diagnostic laparoscopy considered as control group. They had no evidence of pathologic uterine disorder. All of the enrolled patients suffering endometriosis were in the stage III or IV of disease, according to the revised American Society for Reproductive Medicine (rASRM) classification [27]. All endometrial samples were collected during proliferative phase of menstrual cycle. Control and eutopic endometrial samples (12 control and 12 eutopic samples) were obtained using pipelle. Ectopic samples (12 ectopic tissues) were collected during laparoscopy. After collection of endometrial samples (total 36 samples), the tissues were immediately transferred into two separate cryovials. One cryovial containing RNA later solution used to study gene expression and the other without RNA later solution for epigenetic evaluations. All samples were stored at − 80 °C until performing the analysis.

This study was approved by the Institutional Ethics Committee of Royan Institute (code: IR.ACECR.ROYAN.REC.1398.95). All enrolled women signed the informed consent form before tissue samples collection.

RNA extraction and cDNA synthesis

Endometrial tissues were removed from RNA later and then homogenized in 1 ml TRIzol reagent (Kiazol, Iran). Total RNA extraction was done using TRI reagent protocol according to the manufacturer instruction. It was subsequently treated with DNaseI (Takara, Japan) to remove genomic DNA contamination. Concentration and purity of RNA samples were assessed by Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) synthesis was performed using TaqMan reverse transcription kit (Takara, Japan), according to the manufacturer’s instruction.

Quantitative real time polymerase chain reaction (qRT-PCR)

Gene expression assessment was carried out by quantitative real time polymerase chain reaction (qRT-PCR) using the Step-One RT-PCR system (AB Applied Biosystems, USA) and with the primers designed for VEGF, IGF1, IGF2, H19 (the primers listed in Table 1). Human glyceraldehyde dehydrogenase (GAPDH) was used as housekeeping gene. Gene expressions were calculated according to the 2−ΔΔCT algorithm, by normalizing their expression to the GAPDH, as an internal standard.

Table 1 Primer pairs used in this study

Chromatin immunoprecipitation (ChIP) assay

Chromatin immunoprecipitation (ChIP) experiments were performed by using the Orange ChIP kit (Diagenode, Belgium), according to the manufacturer’s instructions. Briefly, cross-linking between DNA and protein was fixed in homogenized endometrial tissues by adding formaldehyde (37%; Sigma, USA). Next, by adding glycine, the cross-linking reaction induced by formaldehyde was quenched. Sonication was used to fragment chromatin to an average DNA fragment size of 200–600 bp using the Bioruptor Sonication System (Diagenode, UCD 200 Bioruptor). One percent of the sheared chromatin was saved, as control input DNA (without adding any antibody), while the rest was incubated with anti-MeCP2 antibody (Abcam, UK) for immunoprecipitation. Real time quantitative PCR (qPCR) was used to analyze level of DNA methylation modifications of H19-DMR region with specific primer sets (Table 1). The primers were designed to amplify two different regions of H19-DMR. Data is reported based on the fold enrichment of different immunoprecipitated DNA relative to 1/100 dilution of input chromatin. The % input was determined using the following formula: % input = 2(CtP inputP – CtP ChIPP) B B × Fd × 100%.

Statistical analysis

Data analyses were performed using SPSS 16 software. One-way analysis of variance (ANOVA) test and non-parametric Kruskal–Wallis test were used when distribution of the values was normal and not normal, respectively. Values were expressed as mean ± standard error of mean (SEM). A p-value less than 0.05 was taken into consideration as statistical significant.

Results

Expression analysis of VEGF, IGF1, IGF2 and H19 genes

To determine relative expression levels of the VEGF, IGF1, IGF2 and H19 genes in eutopic, ectopic and control groups, qRT-PCR was performed during proliferative phase of menstrual cycle. According to Fig. 1A, gene expression level of VEGF was increased in eutopic tissue, in comparison with control and ectopic samples. There was a decrease in VEGF level in ectopic endometrium versus eutopic samples and control group. Although these differences were not statistically significant (p > 0.05; Fig. 1A). Expression levels of IGF1 and IGF2 were decreased in ectopic endometriotic lesions, compared to the eutopic and control groups. In addition, expression levels of these two genes were lower in eutopic samples compared to control group, although the differences were not significant (p > 0.05; Fig. 1B and C). As shown in Fig. 1D, expression of H19 was decreased in eutopic and ectopic endometrial lesions of endometriosis group, in comparison with the control group. Statistical analyses showed that decrease in ectopic samples was significant compared to control group, while it was marginally significant compared to the eutopic samples (p = 0.01 and p = 0.056, respectively). However, there was no significant difference in expression profile of H19 between eutopic and control groups (p > 0.05; Fig. 1D).

Fig. 1
figure 1

Relative mRNA expression levels of VEGF, IGF1, IGF2 and H19 genes in eutopic and ectopic tissue samples (n = 12 in each group) from endometriosis patients vs. control group (n = 12), in proliferative phase. The results are expressed as 2−ΔΔCT (mean ± SEM). Expression of H19 significantly decreased in ectopic samples compared to control group. Different letters indicate significant difference between the eutopic, ectopic and control groups in p < 0.05

DNA methylation of the DMR region of H19 gene

In order to evaluate DNA methylation on DMR of H19, MeCP2 incorporation on this region was investigated in eutopic, ectopic and normal tissues by ChIP assay. Two sub-regions (region I and region II in Fig. 2A) were analyzed within H19-DMR. Region I contains the sixth CTCF-binding site. It has been proposed that this site has a key regulatory domain for the imprinted expression of H19 and IGF2 genes [28]. ChIP analysis revealed that MeCP2 incorporation at the first DMR region of H19 gene was not significantly different between the eutopic, ectopic and control samples (p > 0.05; Fig. 2A). Furthermore, a significant hypomethylation at the second DMR region of H19 gene was observed in the eutopic samples compared to the control group (p = 0.02), while this decrease was marginally significant in the ectopic samples versus control samples (p = 0.056). However, there was no significant difference in DNA methylation (MeCP2 incorporation) of the second region of H19-DMR, between the ectopic and eutopic endometrial samples (p > 0.05; Fig. 2B).

Fig. 2
figure 2

DNA methylation profile of H19-DMR regulatory regions by chromatin immunoprecipitation (ChIP) assay. A The regions amplified by qPCR are shown by arrows, and nucleotide numbers are relative to the transcription start site. Different letters indicate significant differences between the groups (p < 0.05). Incorporation of MeCP2 on the regulatory regions of B region I, C region II in endometriosis and control groups. The data showed significant hypo-methylation in region II in eutopic group compared to control group. There was no significant difference in the incorporation of MeCP2 in region I between the eutopic, ectopic and control samples. The results are expressed relative to a 1/100 dilution of input chromatin (mean ± SEM)

Discussion

Despite many studies, the exact cause of endometriosis is yet unclear. Currently, several evidences showed that neo-angiogenesis was a major element in implantation and survival of endometrial lesions outside the uterine cavity. So, VEGF may have important roles in the pathogenesis of endometriosis [29, 30]. In the present study, gene expression of VEGF was increased in the eutopic endometrial tissues of women with endometriosis compared to the controls. Moreover, expression of this gene was lower in the ectopic lesions versus eutopic and control endometrial samples. This finding was consistent with Cosín et al. study showing that mRNA expression of VEGF was increased in the eutopic endometrium of endometriosis compared to the controls in proliferative phase [31]. Rashidi et al. showed that there was no significant difference in VEGF mRNA expression between endometriosis and control groups. However, it was reported that mRNA expression of VEGF in the case group was higher in the secretory phase than in the proliferative phase [32]. In 2018, Zhang et al. showed that expression of VEGF was significantly increased in both serum and endometrial tissues and it is correlated with the R-AFS (American Fertility Society classification) stage [33]. In relation to endometriotic lesions, Takearaet et al. indicated that expression of VEGF mRNA was increased in the endometriotic tissue (early stages) compared to eutopic endometrium [34]. However, in the present study, we did not detect any increase in VEGF mRNA expression in ovarian endometriomas (stages III–IV). In agreement with our study, a reduction of VEGF level was reported in ovarian endometrioma compared to the eutopic endometrium in endometriosis [35, 36]. Additionally, decreased angiogenic activity of endometriotic lesions was observed in the advanced stages [37, 38]. In addition, Bourlev et al. showed that in ectopic endometrium, samples with high proliferative activity in glandular epithelial cells indicated greater expression of VEGF-A in stromal and epithelial cells than samples with low proliferative activity [39]. Results suggested that overexpression of VEGF in the eutopic endometrium of endometriosis could represent higher angiogenic activity, which might contribute to the increased ability of endometrial cell implantation at the ectopic sites. Insulin-like growth factor (IGF) axis appears to be involved in regulation of endometrial cell proliferation and differentiation [40]. LncRNA H19 could regulate IGF‐1 signaling pathway and consequently proliferation as well as apoptosis of endometrial stromal cells [41]. Previous studies reported close relationship between H19 and IGF1 signaling pathway. H19 can also recruit epigenetic modifiers, acting as a guide to suppress gene expression [42, 43]. Our results showed that women with endometriosis had decreased IGF1 expression as well as decrease in the level of IGF2 expression in the eutopic and ectopic endometrium of endometriosis group compared to the controls. In agreement with our study, Sbracia et al. showed decreased IGF2 expression level in the eutopic and ectopic epithelial endometrial cells of endometriotic in comparison with control. In addition, IGF1 was downregulated in the eutopic endometrium of women with endometriosis compared to the control endometrial samples, whereas increased IGF1 expression in fibrotic peritoneal adhesions was observed [40]. Milingos et al. reported that a significant decrease in IGF1 expression in endometriotic cyst in comparison to eutopic endometrium of women with endometriosis [44]. Our results were inconsistent with Arablou et al. study demonstrating significantly higher IGF1 expression in the ectopic endometrial stromal cells (EESCs) compared to EuESCs and CESCs [45]. This apparent difference is probably due to the cell culture. Because in vitro manipulation of isolated stromal cells and lack of in vivo crosstalk with other cells may play a role in the altered expression of IGF1 in cell culture.

According to the present results, H19 gene expression was significantly lower in the ectopic lesion compared to the eutopic and control endometrium. Similar to our results, Ghazal et al. detected that H19 expression was decreased in the eutopic endometrium of patients with endometriosis in comparison with the controls [26]. Furthermore, in 2019 Liu et al. showed that lncRNA H19 was downregulated in mononuclear cells from peritoneal fluid (PFMCs) of patients with endometriosis [46]. But a few studies exhibited that lncRNA H19 expression in the ectopic and eutopic endometrial tissues of endometriosis was significantly higher than the normal endometrium [47,48,49]. This discrepancy is likely the result of differences in race and maybe stage of disease. So, more genetic investigations are needed. In addition our results were based on in vivo experiments, so further in vitro studies are required.

In the present study, decreased expression of IGF1 could be due to decreased H19 expression level. It was determined that IGF1, mediated via IGF1 receptor (IGF1R), activated PI3K/AKT and Ras/Raf/MAPK signal transduction pathways, which enhanced uterine cell proliferation [7]. During proliferative phase of the menstrual cycle, estradiol induces H19 expression in the endometrium. Uprising of H19 causes increased level of Igf1r protein. This increase leads to upregulating IGF1 signaling with an increased proliferation of endometrial stromal cells [26]. This pathway seems to be changed in women with endometriosis. Therefore, in our study, decreased H19 expression probably reduces expression of IGF1 and thus decreased IGF1 signaling, leading to reduced stromal cell proliferation rate in endometriosis patients. These alterations in proliferation of endometrial stromal cell may be a potential mechanism for infertility in women with endometriosis. Since endometrioma indicates late stages of endometriosis, reduction of IGF1 expression in cysts is likely related to the disease status. In addition, H19 expression level may be correlated to disease progression and infertility.

In our study, decreased expression of IGF2 could be due to two reasons; epigenetic mechanisms by H19 and reducing methylation in H19/ICR. Earlier studies showed that lncRNA H19 binds methyl-CpG-binding domain protein 1 (MBD1). LncRNA H19–MBD1 complex binds to methylated DNA after which recruits histone lysine methyltransferases (KMTs) to silence these genes via chromatin compaction (H3K9 methylation) [50]. The function of H19-DMR methylation on IGF2 expression, resulting from loss of imprinting of IGF2, was observed in many studies [51, 52]. As previously mentioned, DNA methylation in H19-DMR region is epigenetic alterations which can induce imprinting and affect gene expression. To determine whether reduced IGF2 and H19 gene expressions in endometriosis was because of epigenetic modifications or not, we then analyzed epigenetic alterations of the H19-DMR regulatory regions. Generally, in normal conditions when the ICR is methylated, MeCP2, as part of the methyl-binding protein family (MBDs), binds to methylated CpG dinucleotides. This is the main mechanism through which DNA methylation can suppress transcription of H19 gene and the enhancer region is capable of interacting with IGF2 to promote expression. Conversely, when ICR is not methylated, CTCF binds and allows the same enhancer region to promote H19 expression. Therefore, this region and its methylation are essential for both H19 and IGF2 expression [53, 54].

Our results showed that methylation modifications on the regulatory regions of H19-DMR in region II is consistent with the pattern of IGF2 gene expression. However, expression level of H19 was not associated with methylation changes in regions I and II. Furthermore, incorporation of MeCP2 in region I within DMR region of H19 gene was not significantly different between endometriosis and normal tissues. It has been shown that hypermethylation of CTCF-binding site at the H19/ICR increased expression of IGF2 in ovarian cancer [55]. Alternatively, in esophageal squamous cell carcinoma, H19 CTCF-binding site 6 (CBS6) hyper-methylation correlates with overexpression of IGF2 in patients [56]. In another study, it was shown that alteration of H19/IGF2 expression patterns, due to hypo-methylation of H19-DMR, may play roles in the pathogenesis of pregnancy complications [57]. However, no study was performed on methylation modifications of H19-DMR in endometriosis. Results of the current study showed that methylation modifications of region II of H19-DMR can alter expression level of IGF2, but it has no effect on H19 expression. It is hypothesized that epigenetic modifications of the other regions of H19-DMR can probably affect expression profile of H19 gene.

Conclusion

In conclusion, according to the mentioned results, overexpression of VEGF in the eutopic endometrium could lead to implantation of endometrial fragments in extra uterine cavity. Decreased H19 expression in endometriosis lesions probably decreased IGF1 and IGF2 expression. This pattern implies that the cells of endometriotic tissue possibly undergo an impairment of cellular growth regulation and differentiation. Finally, epigenetic findings of the present study showed that region II of H19-DMR can affect expression profile of IGF2, although more investigations are required to clarify roles of the further epigenetic modifications in this region as well as the studying more target genes for H19.

Availability of data and materials

Supporting and raw data are available upon a reasonable request to the corresponding author.

Abbreviations

VEGF:

Vascular endothelial growth factor

IGF1:

Insulin‐like growth factor 1

IGF2:

Insulin‐like growth factor 2

lncRNAs:

Long noncoding RNAs

MeCP2:

Methyl CpG binding protein 2

DMR:

Differentially methylated region

ICR:

Imprinting control region

ChIP:

Chromatin immunoprecipitation

References

  1. Gordts S, Koninckx P, Brosens I. Pathogenesis of deep endometriosis. Fertil Steril. 2017;108(6):872–85.

    Article  PubMed  Google Scholar 

  2. Gupta S, Agarwal A, Krajcir N, Alvarez JG. Role of oxidative stress in endometriosis. Reprod Biomed. 2006;13(1):126–34.

    Article  CAS  Google Scholar 

  3. Da Broi MG, Ferriani RA, Navarro PA. Ethiopathogenic mechanisms of endometriosis-related infertility. JBRA Assist Reprod. 2019;23(3):273.

    PubMed  PubMed Central  Google Scholar 

  4. Viganò P, Parazzini F, Somigliana E, Vercellini P. Endometriosis: epidemiology and aetiological factors. Best Pract Res Clin Obstet Gynaecol. 2004;18(2):177–200.

    Article  PubMed  Google Scholar 

  5. Laganà AS, Vitale SG, Salmeri FM, Triolo O, Frangež HB, Vrtačnik-Bokal E, Stojanovska L, Apostolopoulos V, Granese R, Sofo V. Unus pro omnibus, omnes pro uno: a novel, evidence-based, unifying theory for the pathogenesis of endometriosis. Med Hypotheses. 2017;1(103):10–20.

    Article  CAS  Google Scholar 

  6. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellvé AR, Efstratiadis A. Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol. 1996;10(7):903–18.

    CAS  PubMed  Google Scholar 

  7. Ivanga M, Labrie Y, Calvo E, Belleau P, Martel C, Luu-The V, Morissette J, Labrie F, Durocher F. Temporal analysis of E2 transcriptional induction of PTP and MKP and downregulation of IGF-I pathway key components in the mouse uterus. Physiol Genomics. 2007;29(1):13–23.

    Article  CAS  PubMed  Google Scholar 

  8. Zhou Y, Zeng C, Li X, Wu PL, Yin L, Yu XL, Zhou YF, Xue Q. IGF-I stimulates ERβ and aromatase expression via IGF1R/PI3K/AKT-mediated transcriptional activation in endometriosis. J Mol Med. 2016;94(8):887–97.

    Article  CAS  PubMed  Google Scholar 

  9. Forster R, Sarginson A, Velichkova A, Hogg C, Dorning A, Horne AW, Saunders PT, Greaves E. Macrophage-derived insulin-like growth factor-1 is a key neurotrophic and nerve-sensitizing factor in pain associated with endometriosis. FASEB J. 2019;33(10):11210–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vu TH, Hoffman AR. Promoter-specific imprinting of the human insulin-like growth factor-II gene. Nature. 1994;371(6499):714–7.

    Article  CAS  PubMed  Google Scholar 

  11. Tang XM, Rossi MJ, Masterson BJ, Chegini N. Insulin-like growth factor I (IGF-I), IGF-I receptors, and IGF binding proteins 1–4 in human uterine tissue: tissue localization and IGF-I action in endometrial stromal and myometrial smooth muscle cells in vitro. Biol Reprod. 1994;50(5):1113–25.

    Article  CAS  PubMed  Google Scholar 

  12. Laschke MW, Giebels C, Menger MD. Vasculogenesis: a new piece of the endometriosis puzzle. Hum Reprod Update. 2011;17(5):628–36.

    Article  CAS  PubMed  Google Scholar 

  13. Melincovici CS, Bosca AB, Susman S, Marginean M, Mihu C, Istrate M, Moldovan IM, Roman AL, Mihu CM. Vascular endothelial growth factor (VEGF)-key factor in normal and pathological angiogenesis. Rom J Morphol Embryol. 2018;59(2):455–67.

    PubMed  Google Scholar 

  14. Di Carlo C, Bonifacio M, Tommaselli GA, Bifulco G, Guerra G, Nappi C. Metalloproteinases, vascular endothelial growth factor, and angiopoietin 1 and 2 in eutopic and ectopic endometrium. Fertil Steril. 2009;91(6):2315–23.

    Article  PubMed  CAS  Google Scholar 

  15. Li S, Hua Y, Jin J, Wang H, Du M, Zhu L, Chu H, Zhang Z, Wang M. Association of genetic variants in lncRNA H19 with risk of colorectal cancer in a Chinese population. Oncotarget. 2016;7(18):25470.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Gabory A, Jammes H, Dandolo L. The H19 locus: role of an imprinted non-coding RNA in growth and development. BioEssays. 2010;32(6):473–80.

    Article  CAS  PubMed  Google Scholar 

  17. Brannan CI, Dees EC, Ingram RS, Tilghman SM. The product of the H19 gene may function as an RNA. Mol Cell Biol. 1990;10(1):28–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Tanos V, Ariel I, Prus D, De-Groot N, Hochberg A. H19 and IGF2 gene expression in human normal, hyperplastic, and malignant endometrium. Int J Gynecol Cancer. 2004;14(3):1–7.

    Article  Google Scholar 

  19. Nasu K, Kawano Y, Tsukamoto Y, Takano M, Takai N, Li H, Furukawa Y, Abe W, Moriyama M, Narahara H. Aberrant DNA methylation status of endometriosis: epigenetics as the pathogenesis, biomarker and therapeutic target. J Obst Gynaecol Res. 2011;37(7):683–95.

    Article  CAS  Google Scholar 

  20. Bogdanović O, Veenstra GJ. DNA methylation and methyl-CpG binding proteins: developmental requirements and function. Chromosoma. 2009;118(5):549–65.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Marášek P, Dzijak R, Studenyak I, Fišerová J, Uličná L, Novák P, Hozák P. Paxillin-dependent regulation of IGF2 and H19 gene cluster expression. J Cell Sci. 2015;128(16):3106–16.

    PubMed  PubMed Central  Google Scholar 

  22. Ulaner GA, Yang Y, Hu JF, Li T, Vu TH, Hoffman AR. CTCF binding at the insulin-like growth factor-II (IGF2)/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues. Endocrinology. 2003;144(10):4420–6.

    Article  CAS  PubMed  Google Scholar 

  23. Nordin M, Bergman D, Halje M, Engström W, Ward A. Epigenetic regulation of the Igf2/H19 gene cluster. Cell Prolif. 2014;47(3):189–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pope C, Mishra S, Russell J, Zhou Q, Zhong XB. Targeting H19, an imprinted long non-coding RNA, in hepatic functions and liver diseases. Diseases. 2017;5(1):11.

    Article  PubMed Central  CAS  Google Scholar 

  25. Korucuoglu U, Biri AA, Konac E, Alp E, Onen IH, Ilhan MN, Turkyilmaz E, Erdem A, Erdem M, Menevse S. Expression of the imprinted IGF2 and H19 genes in the endometrium of cases with unexplained infertility. Eur J Obst Gynecol Reprod Biol. 2010;149(1):77–81.

    Article  CAS  Google Scholar 

  26. Ghazal S, McKinnon B, Zhou J, Mueller M, Men Y, Yang L, Mueller M, Flannery C, Huang Y, Taylor HS. H19 lnc RNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis. EMBO Mol Med. 2015;7(8):996–1003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine revised classification of endometriosis 1996. Fertil Steril. 1997;67:817–21.

    Article  Google Scholar 

  28. Takai D, Gonzales FA, Tsai YC, Thayer MJ, Jones PA. Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer. Hum Mol Genet. 2001;10(23):2619–26.

    Article  CAS  PubMed  Google Scholar 

  29. McLaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update. 2000;6(1):45–55.

    Article  CAS  PubMed  Google Scholar 

  30. Liu S, Xin X, Hua T, Shi R, Chi S, Jin Z, Wang H. Efficacy of anti-VEGF/VEGFR agents on animal models of endometriosis: a systematic review and meta-analysis. PLoS ONE. 2016;11(11):e0166658.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Cosín R, Gilabert-Estellés J, Ramón LA, España F, Gilabert J, Romeu A, Estellés A. Vascular endothelial growth factor polymorphisms (− 460C/T,+ 405G/C, and 936C/T) and endometriosis: their influence on vascular endothelial growth factor expression. Fertil Steril. 2009;92(4):1214–20.

    Article  PubMed  CAS  Google Scholar 

  32. Rashidi BH, Sarhangi N, Aminimoghaddam S, Haghollahi F, Naji T, Amoli MM, Shahrabi-Farahani M. Association of vascular endothelial growth factor (VEGF) Gene polymorphisms and expression with the risk of endometriosis: a case–control study. Mol Biol Rep. 2019;46(3):3445–50.

    Article  CAS  PubMed  Google Scholar 

  33. Zhang F, Liu XL, Wang W, Dong HL, Xia YF, Ruan LP, Liu LP. Expression of MMIF, HIF-1α and VEGF in serum and endometrial tissues of patients with endometriosis. Current Med Sci. 2018;38(3):499–504.

    Article  CAS  Google Scholar 

  34. Takehara M, Ueda M, Yamashita Y, Terai Y, Hung YC, Ueki M. Vascular endothelial growth factor A and C gene expression in endometriosis. Hum Pathol. 2004;35(11):1369–75.

    Article  CAS  PubMed  Google Scholar 

  35. Gilabert-Estellés J, Ramón LA, Espana F, Gilabert J, Vila V, Réganon E, Castelló R, Chirivella M, Estellés A. Expression of angiogenic factors in endometriosis: relationship to fibrinolytic and metalloproteinase systems. Hum Reprod. 2007;22(8):2120–7.

    Article  PubMed  CAS  Google Scholar 

  36. Ramon LA, Braza-Boïls A, Gilabert-Estellés J, Gilabert J, España F, Chirivella M, Estellés A. microRNAs expression in endometriosis and their relation to angiogenic factors. Hum Reprod. 2011;26(5):1082–90.

    Article  CAS  PubMed  Google Scholar 

  37. Nisolle M, Casanas-Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril. 1993;59(3):681–4.

    Article  CAS  PubMed  Google Scholar 

  38. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod (Oxford, England). 1998;13(6):1686–90.

    Article  CAS  Google Scholar 

  39. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction. 2006;132(3):501–9.

    Article  CAS  PubMed  Google Scholar 

  40. Sbracia M, Scarpellini F, Zupi E, Manna C, Marconi D, Romanini C, Alo P, Di Tondo U, Grasso JA. Differential expression of IGF-I and IGF-II in eutopic and ectopic endometria of women with endometriosis and in women without endometriosis. Am J Reprod Immunol. 1997;37(4):326–9.

    Article  CAS  PubMed  Google Scholar 

  41. Lei Q, Pan Q, Li N, Zhou Z, Zhang J, He X, Peng S, Li G, Sidhu K, Chen S, Hua J. H19 regulates the proliferation of bovine male germline stem cells via IGF-1 signaling pathway. J Cell Physiol. 2019;234(1):915–26.

    Article  CAS  Google Scholar 

  42. Ghanipoor-Samami M, Javadmanesh A, Burns BM, Thomsen DA, Nattrass GS, Estrella CA, Kind KL, Hiendleder S. Atlas of tissue-and developmental stage specific gene expression for the bovine insulin-like growth factor (IGF) system. PLoS ONE. 2018;13(7):e0200466.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Hu S, Zheng J, Du Z, Wu G. Knock down of lncRNA H19 promotes axon sprouting and functional recovery after cerebral ischemic stroke. Brain Res. 2020;1732:146681.

    Article  CAS  PubMed  Google Scholar 

  44. Milingos D, Katopodis H, Milingos S, Protopapas A, Creatsas G, Michalas S, Antsaklis A, Koutsilieris M. Insulin-like growth factor-1 isoform mRNA expression in women with endometriosis: eutopic endometrium versus endometriotic cyst. Ann N Y Acad Sci. 2006;1092(1):434–9.

    Article  CAS  PubMed  Google Scholar 

  45. Arablou T, Delbandi AA, Khodaverdi S, Arefi S, Kolahdouz-Mohammadi R, Heidari S, Mohammadi T, Aryaeian N. Resveratrol reduces the expression of insulin-like growth factor-1 and hepatocyte growth factor in stromal cells of women with endometriosis compared with nonendometriotic women. Phytother Res. 2019;33(4):1044–54.

    Article  CAS  PubMed  Google Scholar 

  46. Liu Z, Liu L, Zhong Y, Cai M, Gao J, Tan C, Han X, Guo R, Han L. LncRNA H19 over-expression inhibited Th17 cell differentiation to relieve endometriosis through miR-342-3p/IER3 pathway. Cell Biosci. 2019;9(1):84.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Xu Z, Zhang L, Yu Q, Zhang Y, Yan L, Chen ZJ. The estrogen-regulated lncRNA H19/miR-216a-5p axis alters stromal cell invasion and migration via ACTA2 in endometriosis. Mol Hum Reprod. 2019;25(9):550–61.

    Article  CAS  PubMed  Google Scholar 

  48. Liu S, Qiu J, Tang X, Cui H, Zhang Q, Yang Q. LncRNA-H19 regulates cell proliferation and invasion of ectopic endometrium by targeting ITGB3 via modulating mir-124-3p. Exp Cell Res. 2019;381(2):215–22.

    Article  CAS  PubMed  Google Scholar 

  49. Liu S, Xin W, Tang X, Qiu J, Zhang Y, Hua K. LncRNA H19 Overexpression in Endometriosis and its Utility as a Novel Biomarker for Predicting Recurrence. Reproductive Sciences (Thousand Oaks, Calif.). 2020 May 28.

  50. Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L. H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1. Proc Natl Acad Sci USA. 2013;110(51):20693–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sparago A, Cerrato F, Vernucci M, Ferrero GB, Silengo MC, Riccio A. Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith–Wiedemann syndrome. Nat Genet. 2004;36(9):958–60.

    Article  CAS  PubMed  Google Scholar 

  52. Honda S, Arai Y, Haruta M, Sasaki F, Ohira M, Yamaoka H, Horie H, Nakagawara A, Hiyama E, Todo S, Kaneko Y. Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma. Br J Cancer. 2008;99(11):1891–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao Z, Lobanenkov V, Reik W, Ohlsson R. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA. 2006;103(28):10684–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Li T, Hu JF, Qiu X, Ling J, Chen H, Wang S, Hou A, Vu TH, Hoffman AR. CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop. Mol Cell Biol. 2008;28(20):6473–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Murphy SK, Huang Z, Wen Y, Spillman MA, Whitaker RS, Simel LR, Nichols TD, Marks JR, Berchuck A. Frequent IGF2/H19 domain epigenetic alterations and elevated IGF2 expression in epithelial ovarian cancer. Mol Cancer Res. 2006;4(4):283–92.

    Article  CAS  PubMed  Google Scholar 

  56. Gao T, He B, Pan Y, Gu L, Chen L, Nie Z, Xu Y, Li R, Wang S. H19 DMR methylation correlates to the progression of esophageal squamous cell carcinoma through IGF2 imprinting pathway. Clin Transl Oncol. 2014;16(4):410–7.

    Article  CAS  PubMed  Google Scholar 

  57. Yamaguchi Y, Tayama C, Tomikawa J, Akaishi R, Kamura H, Matsuoka K, Wake N, Minakami H, Kato K, Yamada T, Nakabayashi K. Placenta-specific epimutation at H19-DMR among common pregnancy complications: its frequency and effect on the expression patterns of H19 and IGF2. Clin Epigenet. 2019;11(1):113.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Authors would like to convey their sincerest gratitude to their colleagues at both University of Isfahan and Royan Institute.

Funding

This study was supported partly by a research budget from Research Department of University of Isfahan awarded to K. G. (Supervisor) in support of S. K. for obtaining her Ph.D degree from the University of Isfahan.

Author information

Authors and Affiliations

Authors

Contributions

Study design was performed by S.K., E.A., and M.S. Data analysis and experiments were done by S.K., E.A., F.G. Interpretations of data were performed with S.K., E.A., F.G., M.S., and K.G. Manuscript writing was performed by S.K., M.S. and K.G. All authors have read and approved the manuscript.

Corresponding authors

Correspondence to Maryam Shahhoseini or Kamran Ghaedi.

Ethics declarations

Ethics approval and consent to participate

All protocols to use the Human samples were reviewed and confirmed by Ethics committee of Royan Institute (IR.ACECR.ROYAN.REC.1398.95).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kamrani, S., Amirchaghmaghi, E., Ghaffari, F. et al. Altered gene expression of VEGF, IGFs and H19 lncRNA and epigenetic profile of H19-DMR region in endometrial tissues of women with endometriosis. Reprod Health 19, 100 (2022). https://doi.org/10.1186/s12978-022-01406-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12978-022-01406-w

Keywords