Background
Endometriosis is a chronic, benign gynaecological disorder characterized by the presence of endometrial cells at extrauterine sites and associated with chronic pain and infertility. This disease is a highly prevalent disease, presenting in 10–15% of reproductive age women and approximately 25 to 50% of infertile women [
1,
2]. Endometriosis has a severe impact on socioeconomics and the quality of life of patients [
3]. Endometriosis is classified into minimal (I), mild (II), moderate (III) and severe (IV) stages [
4]. The incidence of minimal or mild endometriosis is more frequent than advanced endometriosis. Minimal or mild endometriosis is peritoneal or ovarian endometriotic implants and filmy adhesions on the fallopian tubes or ovaries. The presence of early-stage endometriosis is associated with poor oocyte quality, lower fertilization rate and embryonic developmental competence [
5,
6]. However, no substantial pelvic anatomical changes have been identified. In addition, atypical symptoms or even no symptoms increase the difficulty of diagnosis in minimal or mild endometriosis, which can be delayed on average by 8 to 11 years [
7]. Currently, the sensitivity and specificity of non-invasive diagnostic methods for endometriosis, especially early-stage, are not optimal. The clinical diagnostic indicator cancer antigen 125 (CA125) performs poorly in diagnosing minimal endometriosis, with a sensitivity of 24% [
8]. Therefore, it is urgent to explore novel diagnostic biomarkers.
Metabolomics has emerged as a powerful and reliable tool to identify metabolites and biomarkers present in the biological system under a given physiological condition. Metabolites not only represent the final products of biological regulatory processes but also act as communicators between the information-rich genome and the functional phenotype. In the past few years, several studies identified a list of potential diagnostic candidates in peritoneal fluid, blood and urine from endometriosis patients at different stages of disease and menstrual cycle [
9,
10]. However, potential biomarkers from the eutopic endometrium remain unknown. Therefore, in the current study, ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS) was used to investigate the metabolomic profile of the eutopic endometrium between minimal/mild endometriosis patients and controls. Twelve metabolites were identified as endometriosis-associated biomarkers. The eutopic endometrium metabolomic profile of endometriosis patients was characterized by a significant increase in the concentration of hypoxanthine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lysophosphatidylethanolamine and asparagine. In contrast, the concentration of uric acid was decreased. Our study provides potential biomarkers for the semi-invasive diagnose of endometriosis at minimal-mild stages.
Discussion
In the current study, we applied a UHPLC-ESI-HRMS-based metabolome profiling approach to investigate metabolic changes in the eutopic endometrium samples from endometriosis patients and identified metabolites for early-diagnosed endometriosis. In this regard, 11 metabolites including hypoxanthine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lysophosphatidylethanolamine and asparagine were significantly increased in the endometriosis group, whereas the uric acid level was decreased. The global metabolomics and subsequent multivariate analysis clearly distinguished metabolic changes in the endometriosis patients from those in the matched controls. A combination of three predictors (uric acid, hypoxanthine and lysophosphatidylethanolamine) shows a very good potential for use in diagnosing endometriosis at early stages. However, a study with a larger sample size is needed to obtain stronger evidence and avoid wide confidence intervals in the future.
Endometriosis is a disease characterized by the presence of endometrial glands and stroma at ectopic sites. This gynaecological disease occurs in approximately 10% of women of reproductive age, who present symptoms including dyspareunia, dysmenorrhoea, chronic pelvic pain and subfertility [
16]. Laparoscopy is the gold standard for the diagnosis of endometriosis. However, laparoscopy is an invasive operation with several limitations, such as surgery-associated risks and financial burden [
17]. So far, it has not been able to accurately predict the presence of endometriosis based on non-invasive way. Ultrasound could efficiently detect the presence of ovarian endometriomas, but it is inadequate for the diagnosis of peritoneal endometriosis, deep endometriosis and endometriosis-associated adhesions. CA125 is the most frequently studied biomarker for endometriosis [
18]. However, it may be more beneficial for diagnosing advanced stages (III–IV) compared to early stages (I - II) [
19]. Hirsch et al. showed that CA 125 performs poorly in diagnosing minimal endometriosis, with a sensitivity of 24% [
8]. At present, over 100 potential biomarkers of endometriosis have been reported; however, few markers were useful for the detection of minimal–mild endometriosis [
20]. The diagnosis of endometriosis can be delayed, on average, by 8 to 11 years, which leads to significant symptoms [
7]. Thus, the cost-effectiveness of endometriosis diagnosis and therapy should be urgently improved.
Increasing evidence shows that metabolomics using easily accessible human biosamples has become an effective tool to explore diagnostic biomarkers and investigate disease progression [
21‐
24]. Metabolomics analysis in endometriosis has been performed in peripheral blood, peritoneal fluid, follicular fluid and urine [
25‐
28]. According to the widely accepted theory of retrograde menstruation, the endometrium is the source of ectopic endometriotic foci. Previous studies showed that the eutopic endometrium contributed to the pathogenesis of endometriosis due to the increase of proliferation, migration and invasion of ectopic endometrium [
29‐
33]. In this study, we did not detect a significant difference in patients’ uric acid level in serum. However, uric acid level was reduced by about half in the eutopic endometrium of patients. The differential expression of uric acid in the serum and endometrium indicated that the eutopic endometrium was more representative, stable and similar to ectopic lesions compared to other samples. In addition, we utilized a semi-invasive way of sampling. The pipelle endometrial biopsy can be used without cervical dilatation in the outpatient department and causes minimal discomfort. Thus, metabolomics analysis via pipelle endometrial biopsy is a viable method to explore molecular markers of endometriosis. All the samples were obtained strictly on the third to fifth day after menstrual cessation because we tried to examine samples in the early follicle phase. Although theoretically we should sample on the same day of the menstrual cycle, each patient’s menstrual period and speed of follicle growth varies. We chose this time to collect samples based on hysteroscopic surgical requirements and patient compliance. Unfortunately, we did not collect enough data on patients with advanced endometriosis to analyse because few patients in stages III-IV in our centre had not been exposed to hormonal drugs within 3 months.
Purine metabolites, including inosine, xanthosine, guanosine and hypoxanthine, were significantly upregulated in the eutopic endometrium, whereas uric acid, as the end product of purine metabolism, was remarkably downregulated. This observation indicates that local purine salvage is potentially impaired. Multiple enzymes participate in the purine metabolism process. Among them, purine nucleoside phosphorylase (PNP) is one of the essential enzymes mediating the generation of uric acid from purines. High levels of expression of this enzyme are postulated to reflect extensive programmed cell death during the implantation process [
34,
35]. In addition, pharmacological inhibition of PNP has been demonstrated to be embryo-lethal or teratogenic [
34]. Our data indicated that the accumulation of these purine metabolites and decrease in uric acid level in the eutopic endometrium may be due to suppressed PNP expression. A previous study applied parallel gene expression profiling using high-density oligonucleotide microarrays to investigate the regulation of gene expression in the endometrium [
36], and reduction of PNP expression was found in endometriosis patients, which supports our hypothesis.
Endometriosis has been known to exhibit similar features of malignancy [
37,
38]. Clinical and microscopic examination proved that endometriosis exhibited cancer-like characteristics, demonstrated by uncontrolled growth, cell invasion, neovascularization and apoptosis [
39]. Almost all of the amino acids have been reported to be upregulated in carcinoma tissues in previous studies [
40]. In cancer cells, a high energy demand leads to the alteration of biochemistry including citric acid cycle dysfunction [
41]. Therefore, alternative routes of carbon backbone delivery are required. The increased ectopic endometrium levels of L-arginine, L-tyrosine, leucine, lysine and asparagine observed in the present study might be caused by the alteration of energy metabolism and high turnover of structural protein. These observations are in agreement with a study carried out on serum samples of endometriosis [
42] and metabolic alterations observed in oesophageal cancer patients [
43,
44].