Background
Endometriosis is a common chronic gynecological disease characterized by the presence of endometrial gland and stroma outside the uterine cavity, affecting approximately 10% of reproductive age women [
1,
2]. The common clinical symptoms include pelvic pain, heavy menstrual bleeding, pelvic adhesion, bloating and fatigue. Notably, the prevalence of endometriosis is 0.5% to 5% in fertile women and 25% to 40% in infertile women [
3], suggesting infertility as one possible consequence of endometriosis. To date, the implantation theory is widely accepted, stating that endometrial tissues pass through the fallopian tube, then attach and grow on pelvic tissue. However, this hypothesis cannot explain the existence of endometriosis outside the pelvis or how endometriosis progresses and invades other tissues. Additional factors such as genetic or immune differences have been suggested as possible contributors to trigger the formation of endometriosis [
4‐
6]. Family history and genome-wide linkage studies also support genetic predisposition during the development of endometriosis [
7‐
10]. These studies provide molecular evidence demonstrating endometriosis as a genetic disease, and it is desirable to explore more genetic variations associated with endometriosis.
Similarly to malignant diseases, extensive growth of endometrial cells on the peritoneal surface and invasion of the pelvic organ are very common during the development of endometriosis. This process is frequently associated with several mechanisms involved in angiogenesis and cellular adhesion. In fact, women who have endometriosis appear to be more at risk of developing several different kinds of ovarian cancers [
8,
11‐
13]. An epidemiological study showed that the prevalence rates of endometriosis in patients with endometrioid and clear cell ovarian carcinoma are 19 and 35.9, respectively [
14]. These findings suggest that endometriosis and certain types of ovarian cancer may share several common genetic alterations during pathogenesis. Genes that regulate cell mobility and invasion in ovarian cancers are therefore possible candidates for playing roles in endometriosis.
Mucins are high-molecular-weight glycoproteins with the function of protecting and lubricating the epithelial surface of respiratory, gastrointestinal and reproductive tracts [
15]. Among the mucin proteins, mucin 4 (MUC4) and mucin 1 (MUC1) are the major ones expressed in the endometrial epithelium [
16,
17]. In cancer studies, these two mucins have been shown to be aberrantly expressed in various malignancies and have been validated as novel targets for cancer diagnosis and therapy [
18‐
20]. Distinct from MUC1, the extracellular domain of MUC4 can interact with human epidermal growth factor receptor 2 (HER2) on the cell surface and modulate downstream cell growth signaling by stabilizing and/or enhancing the activity of cell growth receptor complexes [
18,
21,
22]. Consequently, changes of cytoarchitectures and cellular signaling may lead to the increase of cell mobility and tumor cell invasion.
The above findings provide us with clues to hypothesize that genetic variations in the extracellular domain of MUC4, especially those resulting in amino acid substitutions, may play roles involved in the development of endometriosis. With endometriosis as a possible cause of infertility in women, we also would like to study the association of MUC4 single-nucleotide polymorphisms (SNPs) with susceptibility to endometriosis-related infertility.
Discussion
Previous studies have shown polymorphism of cytokines and adhesion molecules which were associated with the pathogenesis of endometriosis [
38,
39]. To the best of our knowledge, however, no other study to date has investigated the possible association of
MUC4 and endometriosis. The purpose of this study was to evaluate whether genetic variations in
MUC4 associate with endometriosis in the Taiwanese population. Our data prove the association of
MUC4 polymorphisms with advanced stages of endometriosis and the related infertility. Since the extracellular domain of MUC4 is critical for HER2 interaction and cell invasiveness, these defined SNPs located in putative functional domains of MUC4 may play important roles during endometriosis development and progression.
The development of endometriosis and certain types of ovarian cancer share several similar clinical features. For example, endometriosis could progressively invade pelvic viscera, resulting in adhesion, and could recur after medical treatment or surgery. Because of the functions involved in the acquisition of adhesion ligands or receptors and the loss of antiadhesion, proteins such as MUC4 and MUC1, the two major mucins present in endometrial epithelium, thus become suspect in endometriosis development [
16,
17]. In addition to gene overexpression, genetic variations in
MUC1 have also been reported as risk factors contributing to cell mobility and the severity of cancer [
40‐
43]. However, the influence of
MUC4 genetic variations on cell behavior remains unclear. In this study, Pro4135Ser (rs2688513) and Ala4693Ser (2246901) substitutions in the putative functional domains of MUC4 were found to be associated with advanced stages of endometriosis. Since MUC4 is an emerging target for ovarian cancer [
18,
19,
44,
45], our study provides a new direction from which to address the roles of MUC4 in the development of gynecological disorders.
To study the genetic effects of mucin proteins by SNPs, the major interest focuses on the variable number of tandem repeat (VNTR) polymorphisms, which result in different-sized gene transcripts. For examples,
MUC1 variations in the VNTR domain have been found to play roles in regulating
Helicobacter pylori binding to gastric cells [
46]. Other studies have also concluded that VNTR polymorphisms can influence T-antigen presentation and the local immune responses, which consequently have potential effects on gastric cancer development [
47,
48]. With regard to
MUC4, a high degree of polymorphism in the VNTR domain was observed in human tissues, including the endometrial epithelium [
49,
50]. However, the different-sized
MUC4 transcripts did not show association with embryo implantation or cancer development. By contrast, MUC4 can promote cell proliferation and antiapoptotic effects in cancer cells by interacting with HER2 on the cell surface [
18,
19,
22], suggesting the potency of functional domains in the extracellular domain of MUC4. In this study, two SNPs (rs2688513 and rs2246901) that locate in a putative functional loop and the VWFD protein binding domain, respectively, were found to be associated with advanced stages of endometriosis. Further study may clarify whether these amino acid substitutions could change the interaction with HER2 and/or play crucial roles in regulating the cellular activity of the spread of endometriosis.
Endometriosis could cause pelvic adhesion and tubal occlusion that may lead to infertility. However, among patients with endometriosis-related infertility, 50% to 60% of them were diagnosed with minimal or mild endometriosis [
3]. Impaired folliculogenesis, bad oocyte quality and impaired implantation of embryo are therefore considered to be the possible mechanisms for endometriosis-related infertility. Changes in cytokines and growth factors in endometrium, follicular fluid and peritoneal fluid have been suggested as the key players for inducing the above-mentioned phenomena [
5]. Recently, several studies have shown that MUC4 could promote cell migration, change the endometrial environment and create weak points in the epithelium, thus facilitating the failure of embryo implantation [
50,
51]. The Carraway
et al. [
52] study also showed that embryo implantation was associated with downregulation of MUC4 expression in an animal model. In this study, women with a T allele in rs882605 had a lower risk of endometriosis-related infertility, whereas rs2688513 and rs2246901 SNPs did not show any association with the reproductive ability of patients. The rs882605 SNP locates in a putative functional loop within the VNTR domain of MUC4 that may control T-cell antigen presentation and the local immune responses. Our findings may support the view that the regulation of local immunity, rather than uncontrolled cell proliferation, in the endometrium may play a more important role in the development of endometriosis-related infertility.
Our study shows that the T/G genotype at rs882605, as compared with the T/T or G/G genotypes, is unique in patients with endometriosis. So far, our data cannot provide sufficient information to explain why the T/T genotype does not show higher risk of endometriosis than T/G. One reason could be the relatively small study group, while other possibilities could exist. For example, the SNPs analyzed are in tight LD with other unknown allele variants which have an opposite effect. In this case, only individuals with the T/G heterozygous genotype could be observed. Because this is a hospital-based study with a modest sample size, enrollment of a larger cohort based on a population approach could help to elucidate the functional role of MUC4 in endometriosis and the related infertility.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CYYC collected samples, carried out the clinical association study and drafted the manuscript. HWC participated in sample collection and the clinical association study. CMC carried out sample preparation and SNP analyses. CYL participated in SNP analyses. CPC participated in the clinical association study. CHL carried out statistical analyses. WYL carried out sample pretreatment and RNA extraction. JJCS carried out the experimental design and drafted the manuscript. FJT carried out the experimental design and revised the manuscript.