Endometrium metabolomic profiling reveals potential biomarkers for diagnosis of endometriosis at minimal-mild stages

Background: The sensitivity and specificity of non-invasive diagnostic methods for endometriosis, especially at early stages, are not optimal. The clinical diagnostic indicator cancer antigen 125 (CA125) performs poorly in the diagnosis of minimal endometriosis, with a sensitivity of 24%. Therefore, it is urgent to explore novel diagnostic biomarkers. We evaluated the metabolomic profile variation of the eutopic endometrium between minimal-mild endometriosis patients and healthy women by ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS).

Our study comprised 29 patients with laparoscopically confirmed endometriosis at stages I-II and 37 infertile women who underwent diagnostic laparoscopy combined with hysteroscopy from January 2014 to January 2015. Eutopic endometrium samples were collected by pipelle endometrial biopsy. The metabolites were quantified by UHPLC-ESI-HRMS. The best combination of biomarkers was then selected by performing step-wise logistic regression analysis with backward elimination.

Twelve metabolites were identified as endometrios is-associated biomarkers. The eutopic endometrium metabolomic profile of the 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. Metabolites were filtered by step-wise logistic regression with backward elimination, and a model containing uric acid, hypoxanthine, and lysophosphatidylethanolamine was constructed. Receiver-operating characteristic (ROC) analysis confirmed the prognostic value of these parameters for the diagnosis of minimal/mild endometriosis with a sensitivity of 66.7% and a specificity of 90.0%. Conclusions:Metabolomics analysis of the eutopic endometrium in endometriosis was effectively characterized by UHPLC-ESI-HRMS-based metabolomics. Our study supports the importance of purine and amino acid metabolites in the pathophysiology of endometriosis and provides potential biomarkers for semi-invasive diagnosis of early-stage endometriosis.

Endometriosis, Metabolomics, UHPLC-ESI-HRMS, Eutopic endometrium * Correspondence: [email protected] †Equal contributors 2School of Pharmaceutical Sciences in Sun Yat-sen University, 132# Waihuandong Road, Guangzhou, University City, Guangzhou 510006, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Li et al. Reproductive Biology and Endocrinology (2018) 16:42 https://doi.org/10.1186/s12958-018-0360-z

Endometriosis is a chronic, benign gynaecological dis- order 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]. Endo- metriosis has a severe impact on socioeconomics and the quality of life of patients [ 3]. Endometriosis is classi- fied into minimal (I), mild (II), moderate (III) and severe (IV) stages [ 4]. The incidence of minimal or mild endo- metriosis is more frequent than advanced endometriosis. Minimal or mild endometriosis is peritoneal or ovarian endometriotic implants and filmy adhesions on the fallo- pian tubes or ovaries. The presence of early-stage endo- metriosis is associated with poor oocyte quality, lower fertilization rate and embryonic developmental compe- tence [ 5, 6]. However, no substantial pelvic anatomical changes have been identified. In addition, atypical symp- toms 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

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 condi- tion. Metabolites not only represent the final products of biological regulatory processes but also act as communi- cators between the information-rich genome and the functional phenotype. In the past few years, several stud- ies 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 chro- matography coupled with ele ctrospray ionization high- resolution mass spectrometry (UHPLC-ESI-HRMS) was used to investigate the metabolomic profile of the euto- pic endometrium between minimal/mild endometriosis patients and controls. Twelve metabolites were identi- fied as endometriosis-associated biomarkers. The euto- pic 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, guano- sine, xanthosine, lysophosphatidylethanolamine and as- paragine. 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.

Subject selection Patient recruitment was carried out at the Sixth Hospital of Sun Yat-sen University, and analysis of the endomet- rium metabolomic profiles was performed at the School of Pharmaceutical Sciences at Sun Yat-sen University. Eutopic endometrium was collected from 68 volunteers (21–38 years old, body mass index less than 30 kg/m 2) from January 2014 to January 2015 who underwent diagnostic laparoscopy combined with hysteroscopy because of infertility. Clinical diagnosis and classification of subjects were performed through laparoscopic surgery to visually confirm the presence of endometriotic lesions. Surgery was carried out on the third to fifth day after menstrual cessation. All participants had regular menstrual cycles (between 21 and 35 days) without hormonal treatment or use of an intrauterine device in the 3 months before sample collection. Endometrial tissues were obtained via Pipelle biopsy during surgery on the 3rd-5th day after the end of their menstrual bleeding. The severity of endometriosis was determined according to the American Society of Reproductive Medicine revised system [ 4]. Patients diagnosed with endometrial polyp, endometritis, submucous myoma or hydrosalpinx should be excluded after confirmation with hysteroscopy combined with laparoscopy and further confirmation by histology. Two volunteers diagnosed by hysteroscopy with endometrial polyps were excluded. No other pathologies were detected in the 68 volunteers. Three volunteers were highly suspicious for endome- trioma on 2 ultrasounds with more than 3 months inter- val. The mean sizes of the cysts were 8 mm, 7 mm and 8 mm, which were confirmed during surgery. Clinical information associated with each sample group is sum- marized in Table 1. After collection, specimens were im- mediately placed into microtubes and preserved in liquid nitrogen until analysis. This study received the ap- proval of the institutional review board, and all patients gave their written informed consent (approval number: G2012021). Sample preparation for metabolomics Endometrial tissues were obtained from 37 healthy women (Control) and 29 women with endometriosis. Sample prep- aration was performed according to a previous report with slight modifications [ 11]. Briefly, 400 μL of 50% chilled methanol was added to 20 mg of tissue sections in tubes containing ceramic beads for homogenization by using a Precellys 24 homogenizer (Bertin, France). The supernatant was transferred into a fresh tube, and 800 μL of chilled 100% acetonitrile was added to precipitate the protein. Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 2 of 10 Samples were centrifuged at 18000×g at 4 °C for 15 min. A total of 500 μL of supernatant was transferred to a fresh tube and dried under vacuum. Samples were re-suspended in 200 μL of 70% acetonitrile for hydrophobic interaction li- quid chromatography (HILIC) mode or in 35% acetonitrile for reversed-phase liquid chromatography (RPLC) mode and then centrifuged at 18000×g at 4 °C for 5 min. Finally, 5 μL of supernatant was transferred to a UPLC vial and injected for UHPLC-ESI-HRMS analysis. The quality con- trol (QC) samples comprised 5 μL of each sample, repre- senting a universal set of metabolites for this study. In addition, blank samples were 70% acetonitrile or 35% acetonitrile. UHPLC-ESI-HRMS measurements of endometrial tissues According to our previously reported method [ 12], chro- matography was performed using an Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) coupled to a QE x a c t i v e™ benchtop Orbitrap high-resolution mass spec- trometer (Thermo Fisher Scientific, San Jose, CA). For the HILIC mode, an Atlantis Silica HILIC 3 μmc o l u m n (100 mm × 2.1 mm, Waters, Milford, MA, USA), total run time 30 min, was employed. Solvent A was 95% acetonitrile containing 10 mM ammonium formate and 0.1% formic acid, and solvent B was 10 mM ammonium formate and 0.1% formic acid in 50% acetonitrile. The linear gradient used was as follows: holding in 100% A for 0 –1 min, increasing to 100% B linearly for 20 min and washing the column for the next 4.9 min, then returning to 100% A until 30 min for column equilibration with a flow rate of 0.3 mL/min. For the RPLC mode, samples were injected onto an Xterra MS C18 5 μm column (100 mm × 2.1 mm, Waters, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid in water (A) and 100% acetonitrile (B). Table 1 Characteristics of participants for endometriosis patients and controls Endometriosis patients (n = 29) Control group (n = 37) P Age (years) 29.69 ± 3.19 29.74 ± 3.43 0.9543 BMI (kg/m2) 21.04 ± 2.08 21.89 ± 3.22 0.2916 AMH (ng/ml) 4.35 ± 2.74 6.4 ± 4.68 0.0661 Uric acid(μmol/l) 271.2 ± 67.38 289.1 ± 63.15 0.3018 The day of sampling 8.667 ± 0.2108 8.45 ± 0.3033 0.5576 The length of menstruation 5.19 ± 0.3209 5 ± 0.3839 0.7044 Endometriosis stage I stage 19 N/A II stage 10 N/A Ovarian endometriomas 3 N/A Fig. 1 Metabolomic analysis of endometrial tissues from patients with endometriosis ( n = 29, blue diamonds) and healthy controls ( n = 37, red diamonds) under positive ionization mode. Score scatter plots for HILIC ( a) and RPLC ( b) modes and OPLS-DA loadings S-plots for HILIC ( c) and RPLC (d) modes. The major ions are labelled in the S-plot Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 3 of 10 The flow rate was kept at 0.3 mL/min during a 22-min run with the following gradient: 100% A for 2 min to 52% A at 4 min to 30% A at 11 min to 25% A at 14 min and kept at 100% B from 16 min to 17 min and 100% A from 18 min to 22 min. The column temperature was kept at 40 °C. Mass spectrometry was performed with an electro- spray ionization source both in positive and negative ionization modes under the following conditions: the spray voltage was 3.5 kV. The capillary and aux gas heater temperature were 300 °C and 350 °C, respectively. Nitro- gen was used as sheath gas (40 arbitrary) and auxiliary gas (10 arbitrary). Data were acquired from 80 to 900 mass- to-charge (m/z) for mass scanning, and the step collision energy 15, 30, 45 eV was used for MS/MS fragmentation of ions. QC samples were injected intermittently to account for the reproducibility and stability of the UHPLC-ESI-HRMS data [13]. Data analysis The mass spectra data were pre-processed by SIEVE 2.2 (Thermo Fisher Scientific, San Jose, CA) to remove the

and generate a multivariate data matrix con- taining aligned peak areas with matched m/z and retention times. Then, SIMCA 13.0 software (Umetrics, Kinnelon, NJ) was applied to find the features that were responsible for the discrimination of the groups. An orthogonal partial least squares discriminant analysis (OPLS-DA) was used to maximize the group discrimination. The candidate markers were selected by examining the S-plot based on the variable importance (VIP) value, which was more than 1.0. The identification of the metabolites was confirmed by compari- sons of fragmentation spectra and m/z through three main online databases: Metlin (http://metlin.scripps.edu), HMDB (http://www.hmdb.ca/ ), and mzcloud ( https://www. mzcloud.org/ )[ 14, 15]. To assess the strength of as- sociation between individual metabolites and minimal/ mild endometriosis, a step-wise logistic regression analysis with backward elimination was used to establish a model and filter crucial metabolites. The receiver op- erating characteristic (ROC) curve was plotted, and the area under the curve (AUC) was calculated. The optimal point on the ROC curve provided the best trade-off between sensitivity and specificity. Statistical testing was carried out by SPSS 19.0 software (IBM Analytics, USA). Data were assessed for normality of distribution using the Shapiro –Wilk test first. Un- paired Student ’s t-test or the non-parametric Mann – Whitney U-test was evaluated with a 95% confidence level for statistical analysis between the two groups. False discovery rate (FDR) control was performed by the SAS PROC MULTITEST with the FDR option Fig. 2 Metabolomic analysis of endometrial tissues from patients with endometriosis ( n = 29, blue diamonds) and healthy controls ( n = 37, red diamonds) under negative ionization mode. Score scatter plots for HILIC ( a) and RPLC ( b) modes and OPLS-DA loadings S-plot for HILIC ( c) and RPLC (d) modes. The major ions are labelled in the S-plot Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 4 of 10 (SAS Inst, Cary, North Carolina, USA). P-values less than 0.05 were considered statistically significant while controlling FDR at 0.05.

Characteristics of participants with endometriosis and controls Clinical information associated with each sample group is summarized in Table 1. A total of 66 volunteers were recruited in this study. Twenty-nine patients had laparo- scopically confirmed endometriosis, staged as minimal (n =1 9 )a n dm i l d(n = 10). Three patients had a laparoscop- ically documented presence of ovarian endometrioma. All of the endometriomas were histologically confirmed. The mean sizes of all the cysts were less than 1 cm. Age, BMI, menstrual cycle, AMH and uric acid in serum were com- parable between the two groups ( P > 0.05). Both the mean day of sampling and the length of menstruation were not significantly different between the endometriosis patients and the control group. No volunteer had a history of smok- ing in this study. Multivariate statistical analysis of difference between the endometriosis and control groups The alignment of all the features in all samples gener- ated a data matrix by SIEVE 2.2 software with an abun- dance of 5388 features under HILIC mode and 3424 features under RPLC mode. To compare the overall vari- ation of metabolic profiles between the endometriosis patients and healthy controls, a classification model was built by the supervised OPLS-DA, which revealed a clear separation between the two groups (Figs. 1a, b and 2a, b ). The model also showed that samples from humans had great individual differences. An OPLS-DA loadings S-plot was performed to highlight significantly different variables in the two groups (Figs. 1c, d and 2c, d). Each point repre- sented a detected ion (variables). The further away from the plot origin an ion point lies, the more the ion contrib- utes to the difference between the two study groups. Therefore, variables plotted at the top or bottom were changed most significantly. Metabolite features of interest were selected by a VIP value > 1.0. With such a strategy, 450 variables from the HILIC mode results and 469 Fig. 3 Identified metabolites with increasing contributions to the difference in metabolomic profiles between the two groups based on VIP scores Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 5 of 10 variables from the RPLC mode results were considered to have impact on the model. Identification of detected metabolites The m/z of the selected variables and their MS/MS frag- mentation spectra were used for comparison with com- pounds annotated in the online databases. Finally, 27 metabolites from positive and negative ionization modes were uniquely identified on the basis of exact mass and retention time. Among them, levels of 12 metabolites cor- responding to high variable importance (VIP > 1) (Fig. 3) were different between the endometriosis and control groups ( P < 0.05). In addition, their detailed information is summarized in Table 2 and labelled in the S-plot (Figs. 1c, d and 2c, d ). Obviously, levels of hypoxan- thine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lyso- phosphatidylethanolamine and asparagine were higher in the endometriosis group than in the control group, whereas the level of uric acid was higher in the control group (Fig. 4). It is noteworthy that the xanthosine level in the endometriosis group was 2.53-fold higher than that of the control group, while the amount of uric acid was decreased by half, indicating that purine metabol- ism was disturbed in endometriosis patients. After using step-wise multivariate logistic regression analysis with backward elimination, a model with three predic- tors was established, including uric acid, hypoxanthine and lysophosphatidylethanolamine, with a sensitivity of 66.7% (95% CI: 0.417 –0.875) and a specificity of 90.0% (95% CI: 0.600 –1.000). The receiver operating charac- teristic (ROC) curve shows improved effects of adding separate variables to the model. The apparent AUC o ft h eR O Cc u r v ef o rt h em o d e lp r e d i c t i n ge n d o m e t - riosis at the minimal/mild stages was 0.868 (95% CI: 0.774–0.963) (Fig. 5). The combination of three vari- ables led to a curve with significantly better perform- ance and allows very good discrimination between endometriosis patients at early stages and controls.

In the current study, we applied a UHPLC-ESI-HRMS- based metabolome profiling approach to investigate meta- bolic 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 aspara- gine 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 endometri- osis patients from those in the matched controls. A com- bination of three predictors (uric acid, hypoxanthine and lysophosphatidylethanolamine) shows a very good poten- tial for use in diagnosing endometriosis at early stages. Table 2 Summary of the data from the 12 features found in positive and negative ionization modes contributing to the discrimination of endometrial tissues between endometriosis patients and healthy controls m/za tR (min)b Metabolite Molecular formula Adduct Fold change c P valued Adj P valuee HILIC mode 131.0462 10.793 Asparagine C 4H8N2O3 M-H 1.44 0.013 0.0173 132.1019 8.833 Leucine C6H13NO2 M + H 1.68 0.002 0.0120 137.0455 4.431 Hypoxanthine C5H4N4O M + H 1.64 0.033 0.0360 167.0209 5.283 Uric acid C5H4N4O3 M-H 0.54 0.000 0.0053 267.0737 4.539 Inosine C10H12N4O5 M-H 1.58 0.037 0.0370 282.0844 6.017 Guanosine C10H13N5O5 M-H 1.55 0.023 0.0276 283.0685 4.771 Xanthosine C10H12N4O6 M-H 2.53 0.008 0.0137 RPLC mode 147.1125 0.696 Lysine C6H14N2O2 M + H 1.54 0.008 0.0137 175.1186 0.703 L-Arginine C6H14N4O2 M + H 1.41 0.004 0.0137 182.0808 1.091 L-Tyrosine C9H11NO3 M + H 1.47 0.006 0.0137 303.2329 15.245 Omega-3 Arachidonic acid C 20H32O2 M-H 1.57 0.005 0.0137 478.2935 10.770 LPE (18:1(9Z)/0:0) C23H46NO7P M-H 1.20 0.013 0.0173 am/z is the detected mass to charge ratio from LC-MS/MS runs bRetention time in minutes cThe fold change of the endometriosis group vs the control group (a higher ratio indicates a higher level of expression of a compound in the EMS group) dP value is the significance level of the difference between the two groups eP values were adjusted for false discovery rate correction at the significance level of 5% LPE lysophosphatidylethanolamine, PC phosphatidylcholine Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 6 of 10 However, a study with a larger sample size is needed to obtain stronger evidence and avoid wide confidence inter- vals in the future. Endometriosis is a disease characterized by the pres- ence 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 endome- triomas, but it is inadequate for the diagnosis of peritoneal endometriosis, deep endometriosis and endometriosis-as- sociated adhesions. CA125 is the most frequently Fig. 4 Scatter diagram of 12 selected metabolites. Data are expressed as the mean ± SD. * P < 0.05, **P < 0.01, ***P < 0.001, endometriosis patients (EMS, n = 29) vs healthy controls (Control, n = 37) Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 7 of 10 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 aver- age, by 8 to 11 years, which leads to significant symp- toms [ 7]. Thus, the cost-effectiveness of endometriosis d i a g n o s i sa n dt h e r a p ys h o u l db eu r g e n t l yi m p r o v e d . Increasing evidence shows that metabolomics using easily accessible human biosamples has become an ef- fective tool to explore diagnostic biomarkers and investi- gate 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 pa- tients’ uric acid level in serum. However, uric acid level was reduced by about half in the eutopic endometrium of pa- tients. The differential expression of uric acid in the serum and endometrium indicated that the eutopic endometrium was more representative, stable and similar to ectopic le- sions 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 out- patient department and causes minimal discomfort. Thus, metabolomics analysis via pipelle endometrial biopsy is a viable method to explore molecular markers of endometri- osis. 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 theor- etically 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 compli- ance. Unfortunately, we did not collect enough data on pa- tients 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, guano- sine and hypoxanthine, were significantly upregulated in the eutopic endometrium, whereas uric acid, as the end product of purine metabolism, was remarkably downregu- lated. This observation indicates that local purine salvage is Fig. 5 Receiver operating characteristic curves for the model of endometriosis at minimal/mild stages Li et al. Reproductive Biology and Endocrinology (2018) 16:42 Page 8 of 10 potentially impaired. Multiple enzymes participate in the purine metabolism process. Among them, purine nucleo- side 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 dea th during the implantation process [34, 35]. In addition, pharmacological inhibition of PNP has been demonstrated to be embryo-lethal or terato- genic [ 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 expres- sion profiling using high-density oligonucleotide microarrays to investigate the regulationof gene expression in the endo- metrium [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 examin- ation proved that endometriosis exhibited cancer-like char- acteristics, 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, al- ternative 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 alter- ations observed in oesophageal cancer patients [ 43, 44].

Metabolomics provides a powerful approach to explore diagnostic biomarkers by analysing changes in metabolic profiles. Overall, this study is the first to demonstrate a comprehensive analysis of metabolic changes in the euto- pic endometrium in endometriosis at early stages. Metabo- lites involved in purine, amino acid and arachidonic acid metabolic pathways could be potential biomarkers for early diagnosis of endometriosis. These findings provide poten- tial biomarkers for semi-invasive diagnosis of endometri- osis at minimal-mild stages in clinical practice. The implications of these individual metabolites in the patho- physiology and analysis of metabolites in all stages of endo- metriosis have now to be further studied. Funding The authors would like to acknowledge the support from the National Natural Science Foundation of China (No. 81601347, 81503156, 81320108027), Natural Science Foundation of Guangdong Province (No. 2014A030310096) and Public Welfare Research and Capacity Building Fund of Guangdong (No. 2016A020218006). Availability of data and materials The data for this study are available from the corresponding author upon reasonable request. Authors’ contributions JJL conceived of the study, wrote the manuscript and supervised patient recruitment. LHG, HZZ, YG and DSL contributed to the study execution and analysis and interpretation of the data. XG performed data analysis and interpretation. JHS and PC reviewed the manuscript. HCB, MH and XYL supervised patient recruitment, collected and evaluated data, and drafted, edited and approved the final version of this paper for submission. All authors read and approved the final manuscript. Ethics approval and consent to participate This study received approval from the Sixth Affiliated Hospital of Sun Yat-sen University Research Ethics Committee (approval number: G2012021). Competing interests The authors declare that they have no competing interests. Publisher’sN o t e Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1Center of Reproductive Medicine, the Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 2School of Pharmaceutical Sciences in Sun Yat-sen University, 132# Waihuandong Road, Guangzhou, University City, Guangzhou 510006, People ’s Republic of China. 3Pharmacy Department, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 4School of Public Health, Guangdong Pharmaceutical University, Guangzhou, China. Received: 15 January 2018 Accepted: 24 April 2018

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