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Dysregulated Sphingolipid in Endometriosis

Yie Hou Lee, Chin Wen Tan, Abhishek Venkatratnam, Chuen Seng Tan, Liang Cui, Seong Feei Loh, Linda Griffith, Steven R. Tannenbaum, and Jerry Kok Yen Chan Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 BioSym (Y.H.L., C.W.T., A.V., L.G., S.R.T.) and Infectious Diseases Inter-Disciplinary Research Groups (Y.H.L., L.C., S.R.T.), Singapore-MIT Alliance for Research and Technology, Singapore 138602; Saw Swee Hock School of Public Health (C.S.T.), National University of Singapore, Singapore 117597; Department of Reproductive Medicine (S.F.L., J.K.Y.C.), KK Women’s and Children’s Hospital, Singapore 229899; Departments of Biological Engineering (L.G.) and Chemistry (S.R.T.) and Center for Gynepathology Research (L.G.), Massachusetts Institute of Technology, Boston, Massachusetts 02139; Department of Obstetrics and Gynecology (J.K.Y.C.), Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228; and Cancer and Stem Cell Biology Program (J.K.Y.C.), Duke-NUS Medical School, Singapore 169857

Background: In endometriosis, the establishment and subsistence of ectopic lesions outside the endometrium suggest an altered cellular state for pathological hyperplasia. Sphingolipids are bioactive compounds, and their and metabolism modulate a range of cellular pro- cesses including proliferation, migration and . We demonstrate that aberrations in sph- ingolipid metabolism occur in women with endometriosis.

Methods: Targeted mass spectrometry on Ͼ120 sphingolipids were measured in the sera (n ϭ 62), peritoneal fluid (n ϭ 63), and endometrial tissue (n ϭ 14) of women with and without endome- triosis. Quantitative RT-PCR and immunohistochemistry were performed on endometrial tissues determine the expression levels of sphingolipid .

Results: Sphingolipidomics identified the in vivo accumulation of numerous sphingolipids, includ- ing the functionally antagonistic glucosylceramides and in the serum and PF of women with endometriosis. We found upregulation of specific sphingolipid enzymes, namely sphingo- synthase 1 (SMS1), sphingomyelinase 3 (SMPD3), and glucosylceramide synthase (GCS) in the endometrium of endometriotic women with corresponding increased GlcCer, decreased sphingo- myelin levels, and decreased apoptosis in the endometrium.

Conclusions: Our sphingolipidomics approach provided evidence of altered sphingolipid metab- olism flux in serum, peritoneal fluid, and endometrial tissue in women with endometriosis. The results provide new information on how sphingolipids and eutopic endometrium may contribute to the pathophysiology of endometriosis. The results also have implications for the use of sphin- golipids as potential biomarkers. (J Clin Endocrinol Metab 99: E1913–E1921, 2014)

ndometriosis affects an estimated 6% to 10% of tion, and infertility (1). Hereditary components are E women of reproductive age, resulting in debilitating known risk factors in endometriosis (2–4), whereas sus- pelvic pain, dysmenorrhea, dyspareunia, painful defeca- tained can be observed through elevated

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: Cer, ; C1P, Cer-1-phosphate; GalCer, galatocsylceramide; GCS, Printed in U.S.A. GlcCer synthase; GlcCer, glucosylceramide; IHC, immunohistochemistry; LacCer, lactosyl- Copyright © 2014 by the Endocrine Society ceramide; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple Received February 5, 2014. Accepted June 18, 2014. reaction monitoring; m/z, mass to charge ratio; OPLS, orthogonal partial least squares; PC, First Published Online June 24, 2014 phosphatidylcholine; PF, peritoneal fluid; rAFS, revised American Fertility Society; SM, ; SMPD1, SM phosphodiesterase 1; SMS1, SM synthase 1; TUNEL, terminal deoxynucleotide transferase mediated dUTP nick-end labeling.

doi: 10.1210/jc.2014-1340 J Clin Endocrinol Metab, October 2014, 99(10):E1913–E1921 jcem.endojournals.org E1913 E1914 Lee et al Dysregulated Metabolic Processing in Endometriosis J Clin Endocrinol Metab, October 2014, 99(10):E1913–E1921

macrophages and proinflammatory cytokine levels (5, 6). Materials and Methods Treatment modalities for endometriosis are currently in- adequate and empirical, with medical therapy resulting in Patient enrollment and ethics The study population comprising patients presenting with anovulation, a situation unacceptable for patients seeking subfertility in addition to a general gynecological case mix was fertility. Surgical removal or ablation of ectopic endome- recruited in KK Women’s and Children’s Hospital, Singapore. trial implants are currently most effective not only for Women provided written informed consent for collection of alleviation of pain but also for improving fertility. How- samples under Centralized Institutional Research Board ap- ever, both medical and surgical treatments are not without proval (CIRB 2010-167-D). Exclusion criteria included men- struating patients, postmenopausal patients, patients on hor- significant side effects or are unsuitable for couples seek- monal therapy for at least 3 months before laparoscopy, and ing fertility (7) and do not address the issue of disease other confounding diseases such as diabetes, other chronic in- Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 recurrence (8). Therefore, elucidating the mechanisms of flammatory diseases (rheumatoid arthritis, inflammatory bowel enhanced will greatly enhance our under- disease, etc). Serum (n ϭ 62), PF (n ϭ 63), and endometrial ϭ standing of the disease pathobiology and generate novel samples (n 14) were collected from women undergoing lapa- roscopic procedures for suspected endometriosis, infertility, ster- therapeutic targets directed at the pathophysiological ilization procedures, and/or pelvic pain. A careful survey of the mechanisms to ultimately improve the quality of life of uterus, fallopian tubes, ovaries, and the pelvic peritoneum was affected women. made for any evidence of endometriotic deposits, and the severity Although numerous molecular events related to lesion of endometriosis was scored according to the revised American subsistence have been implicated (9), the contribution of Fertility Society (rAFS) classification of endometriosis (21, 22). A total of 38 patients with endometriosis (EMϩ), and 24 women sphingolipid metabolism, in particular ceramide (Cer) me- who did not have endometriosis or have benign gynecological tabolism, has not been explored. Sphingolipids are in- diagnoses (uterine fibroids, benign ovarian cysts, etc) were in- creasingly known to be important bioactive signaling mol- cluded as the control group (EMϪ). For PF analysis, a cohort of 63 subjects, of whom 45 were common to the serum cohort, ecules and are involved in a diverse range of cellular ϩ Ϫ processes. For example, Cer and sphinghoid bases mod- comprised 39 EM subjects and 26 EM subjects. Further de- tails on patient characteristics can be found in Table 1. For check- ulate many apoptosis signaling events, whereas their phos- ing specificity of sphingolipids in relation to endometriosis, se- phorylation or lead to the production of mi- rum from an additional 5 EMϩ and5EMϪ menstruating women togenic factors such as Cer-1-phosphate (C1P) and were collected and analyzed. The phase of the menstrual cycle glucosylceramide (GlcCer). Elevated levels of GlcCer have (proliferative and secretory) was determined according to cycle history of the patients. been documented in many chronic human diseases such as Gaucher’s disease, cancers, type 2 diabetes mellitus, and Sample preparation polycystic kidney disease (10–13). Studies on renal epi- Blood was collected and serum prepared by centrifugation at thelial cells (14), keratinocytes (15, 16) and cancer cell 1200g for 10 minutes and the top layer respun at 3600g for 10 lines (17, 18) further demonstrate the mitogenic properties minutes. The supernatant was carefully removed and transferred of GlcCer. In addition, mice with ␤-glucosidase mutations into 1-mL aliquots. PF was collected from the pouch of Douglas, and 1% vol/vol protease inhibitor was added (Roche), spun at display systemic inflammation (19). Central among the 1000g for 10 minutes and the supernatant transferred to 15-mL GlcCer changes is GlcCer synthase (GCS), the that aliquots. Both sera and PF were stored at Ϫ80°C until use. Uter- catalyzes the glycosylation of Cer, and constitutes the first ine endometrial curettage tissues were irrigated in saline and split committed step for the biosynthesis of higher glycosphin- into 2 parts: one section was processed for formalin-fixed-par- golipids that are also implicated in cellular proliferation affin embedding for histology, and the other section was kept on ice and frozen at Ϫ80°C (within 4 hours of collection). (20). Given the heightened cellular proliferative states as- Sphingolipids were extracted from sera, PF, and tissues using sociated with endometriosis, we hypothesized that dys- a modified Bligh and Dyer method. Briefly, 900 ␮L chloroform- regulation in the sphingolipid may be methanol (1:2, vol/vol) was added to 100 ␮L serum or PF. Al- associated with intrinsic enhanced endometrial cell pro- ternatively, endometrial tissues were incubated in chloroform- liferation, which leads to lesion growth and implantation methanol for 30 minutes and homogenized using TissueLyser LT (QIAGEN) before the addition of 900 ␮L chloroform-methanol at ectopic sites. (1:2, vol/vol). After 20 minutes vortexing and incubation at 4°C, In this study, we used a targeted quantitative liquid ␮ ␮ 300 L chloroform and 300 L ddH2O were added to the mix- chromatography-tandem mass spectrometry (LC-MS/ ture. Sphingolipids were removed from the lower organic phase MS) lipidomics approach to analyze serum, peritoneal after centrifugation at 9000 rpm at 4°C for 2 minutes. Subse- ␮ fluid (PF), and endometrial tissue sphingolipid concentra- quently, 500 L chloroform was added and vortexed at 4°C for 20 minutes, and were removed from the lower organic tions en masse in woman with endometriosis and immu- phase after centrifugation and combined with the previous frac- nohistochemistry (IHC) to understand dysregulation in tion. The extracts were vacuum-dried and stored at Ϫ80°C. sphingolipid metabolism and fluxes. Before LC-ESI-multiple reaction monitoring (MRM) lipid ex- doi: 10.1210/jc.2014-1340 jcem.endojournals.org E1915

Table 1. Summary of Clinical Parameters of Study Cohort for Serum and PF Analysis

Serum PF

P Valuea (26 ؍ EM؊ (n (39 ؍ P Valuea EM؉ (n (24 ؍ EM؊ (n (38 ؍ EM؉ (n Age, y Median 33.5 34.5 0.22 34 35 0.56 Range 22–44 22–47 22–44 22–51 Diagnostic, rAFS grade I–II 11 NA 13 NA III–IV 27 NA 22 NA Cycle phase 0.29 0.44 Proliferative 21 9 20 10 Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 Secretory 17 14 18 15 Ethnicity 0.79 0.95 Chinese 27 18 26 18 Malay 7 0 5 3 Othersb 44 74 Fecundity 0.64 0.43 Fertile 8 6 8 6 Infertile 28 18 29 14 Subfertile 2 0 1 2 Abbreviation: NA, not available. a Two-tailed Mann-Whitney U test for EMϩ and EMϪ; ␹2 and Fisher’s exact tests of association for categorical values. b Other races comprised Burmese, Indians, Indonesians, Filipinos, and Vietnamese.

tracts were reconstituted in 200 or 400 ␮L chloroform-methanol (LC-Si, 2.1 ϫ 250 mm; inner diameter, 5 ␮m; Supelco, Sigma- (1:1, vol/vol). Lipids were analyzed within 2 weeks after Aldrich). A 10.5-minute isocratic elution using the mobile phase

extraction. (CH3CN/CH3OH/CH3COOH, 97/2/1, vol/vol, with 5mM am- monium acetate) at 750 ␮L/min was used to resolve and identify Mass spectrometry-based sphingolipidomics the isomers using their corresponding internal standards (23). Positive ionization-mode LC-MS/MS via MRM on a triple Peaks were integrated, manually inspected, and quantified using quadrupole 6460 with electrospray ionization source (Agilent MassHunter Quantitative software (versions B.03–05; Agilent). Technologies) was used for the quantification of sphingolipids (156 molecular species; Supplemental Table 1). MRM was based Quantitative-RT-PCR on product ion mass to charge ratio (m/z) 264.4 [- Quantitative RT-PCR was performed as previously described ϩ ϩ H2O] and 266.4 [sphinganine-H2O] for Cer, GlcCer, gala- (24). Total RNA was extracted from tissue samples obtained tocsylceramide (GalCer), (LacCer), and C1P ϩ Ϫ ϩ from 5 EM Sev and 5 EM subjects using TRIzol (Bio-Rad) and m/z 184.1 [] for sphingomyelin (SM) and according to the manufacturer’s instructions. Deoxyribonu- phosphatidylcholine (PC). The product ion m/z 184.1 for SM is clease I (Bio-Rad) was used to remove genomic DNA contami- unable to differentiate the N-linked and sphingoid base nation, total RNA concentration was quantified (NanoDrop; but can be assumed to be the predominant sphingosine base Thermo Scientific) and 1 ␮g RNA was reverse-transcribed by (d18:1) in humans. C-18 reversed-phase LC (Zorbax Eclipse using oligo(dT) and the iScript cDNA synthesis kit (Bio-Rad). ϫ ␮ 2.1 50 mm inner diameter, 1.8 m; Agilent Technologies) was Primer sequences for genes Ugcg, Sptl1, Sptl2, Sptl3, Smpd1, ␮ used to separate reconstituted lipids at 400 L/min before en- Smpd2, Smpd3, Sgms1, and Actb are listed in Supplemental Ta- tering the mass spectrometer. The mobile phase consisted of ble 3. PCR was carried out for 95°C for 30 seconds and 40 cycles 5mM ammonium acetate in water (mobile phase A) and 5mM at 95°C for 1 second and 60°C for 10 seconds. Data were nor- ammonium acetate in methanol (mobile phase B). Sphingolipids malized with Actb. were eluted using linear gradients from 60% to 100% B over 2 minutes, maintained at 100% B for 5 minutes, followed by a linear gradient to 60% B over 2 minutes, and held for another 2 Immunohistochemistry minutes before the next injection. The optimized lipid-class de- IHC was performed as previously described (25) with mod- pendent mass spectrometry parameters are shown in Supple- ifications. Proteinase K-induced antigen retrieval (20 ␮L/mL, pH mental Table 2. Non-naturally occurring internal standards, 8.0) was performed by incubating sections for a half-hour. Al- namely Cer d18:1/17:0, GlcCer d18:1/8:0, GalCer d18:1/8:0, ternatively, in preparation for CD10 detection, heat-induced an- LacCer d18:1/12:0, PC 14:0/14:0, SM 12:0, and C1P d18:1/8:0 tigen retrieval was carried out by microwaving slides in citrate (Avanti Polar Lipids), which corresponded to their sphingolipid buffer (10mM citric acid, 0.05% Tween 20, pH 6.0) for 20 min- classes, were verified for their absence and used for quantifica- utes, blocked with 5% BSA/0.5% Tween 20 in PBS, and incu- tion. Further details on optimization of LC-MS/MS parameters bated overnight at 4°C with primary antibodies against SM can be found in Supplemental Figures 1–3. phosphodiesterase 1 (SMPD1) (Abcam), SMPD2 (Abnova), Geometric isomeric monohexosylceramides GlcCer and Gal- SMPD3 (Abnova), palmitoyl transferase (Abcam), GalCer Cer were separately resolved via normal-phase chromatography (Abcam), GCS (Abbiotec), SM synthase 1 (SMS1) (Proteintech), E1916 Lee et al Dysregulated Metabolic Processing in Endometriosis J Clin Endocrinol Metab, October 2014, 99(10):E1913–E1921 and CD10 (Novocastra). Detection of antigens was performed rate estimation. Fisher’s exact test was used considering the rel- with Alexa Fluor 546 or 3,3=-diaminobenzidine for 1 hour at atively smaller sample set. Nonparametric tests were chosen to room temperature. Samples were washed in PBS and stained with reduce the influence of the imputed values unless otherwise Slow Fade Gold Antifade Reagent with 4=,6-diamidino-2-phe- stated. Heatmaps (MeV version 4.6.2) and OPLS (Unscrambler nylindole (Invitrogen) before imaging through confocal micros- X version 10.1) were analyzed and modeled after the normal- copy (FV1000; Olympus). Primary antibodies replaced with ization of data by first centering the data to the median and blocking buffer acted as negative controls. scaling it by division with the SD. Full cross-validation was ap- plied in OPLS to increase model performance and for the calcu- Terminal deoxynucleotide transferase mediated lation of coefficient regression values. dUTP nick-end labeling assay Terminal deoxynucleotide transferase mediated dUTP nick- end labeling (TUNEL) assay was performed using the TUNEL Results Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 apoptosis detection kit (GenScript). Section processing was sim- ilar to IHC except for endogenous peroxidase activity blocking Differential sphingolipidome in endometriosis with 3% H2O2 in methanol. Apoptotic regions were detected Our cohort of 62 subjects used for serum studies and 63 Streptavidin-fluorescein isothiocyanate. Positive and negative subjects for PF studies (Table 1), of which 45 were com- controls were run in parallel to the samples. Negative controls ϩ mon to the serum cohort, were grouped into cases (EM ) were processed by omitting terminal deoxynucleotidyl trans- and controls (EMϪ). EMϩ subjects were further stratified ferase from the TUNEL reaction mixture, whereas positive con- ϩ trols were incubated with deoxyribonuclease before labeling to to mild cases of endometriosis (EM Mild; rAFS I–II) (21) ϩ induce DNA strand degradation. or severe cases (EM Sev; rAFS III–IV). Age, ethnicity, fer- tility, and menstrual phase were not significant parameters Image analysis (Fisher’s exact test, P ϭ .563, .954, .426, and .444, re- Image analysis was performed using ImageJ. Fluorescent in- spectively). By capitalizing on the fast chromatographic tensity was measured for the sections in general as well as for the separation of ultra–high-performance LC and by coupling individual cell types (stromal and glandular epithelial cells). For both cases, the intensities were normalized with values obtained the selectivity of MRM, we were able to simultaneously Ͼ from the samples’ corresponding negative controls. With respect measure 120 sphingolipids at sensitivities of 0.01 to 0.1 to analysis of the individual cell type’s fluorescent intensities, 5 ng/mL to 10 ng/mL (Supplemental Table 4) at interday glands and stromal sites were randomly selected from each sam- coefficient of variation of 6.5% and intraday coefficient of ple for measurement, and the mean value obtained after nor- variation of 9% (Supplemental Figure 3, A–C). Mono- malization was used for comparison. In addition to comparing hexosylceramides GalCer and GlcCer are geometric iso- the signal intensity across the different samples, comparison was ϩ Ϫ mers in which specific species share the same MRM tran- also made between the EM Sev and EM group as a whole. sitions. For example, the 588.5 m/z 3 264.4 m/z Data preprocessing transition is shared by both GalCer and GlcCer d18:1/ Quantification of sphingolipids was performed via Mass- 24:0 (Supplemental Figure 3D). By chiral chromatogra- Hunter Quantitative software (versions B.04 and B.05; Agilent). phy, we are able to separate GalCer and GlcCer and Sphingolipids with a low signal to noise ratio (response Ͻ50) showed that GalCer represented a small fraction, ϳ0.5% were removed because low responses exhibit decreased repro- to 5% of total monohexosylceramides (Supplemental Fig- ducibility and reliability and therefore may be less quantitatively ure 3E), consistent with other reports (26), and therefore accurate. Hence, only sphingolipids that were represented in Ն70% of the study cohort were accepted for further analyses. To only GlcCer was considered for subsequent analyses. implement statistical modeling (orthogonal partial least squares Unbiased serum sphingolipid profiles displayed as a [OPLS]), undetectable sphingolipid levels were assumed as heat map (Figure 1A) showed pattern discrimination in reaching the detection limits of their analytical method and were relation to the disease. The z-plots revealed serum sphin- replaced with a randomly generated number that is between the golipidome alterations in EMϩ patients (z-score range, lowest measurable value and 1/10 of the lowest measureable Ϫ Ϫ3.1 to 5.4) compared with EM (z-score range, Ϫ0.99 to value. ϩ 2.56) and to a larger extent in EM Sev (Figure 1B). There Statistical analysis were widespread elevations of PF and serum sphingolipid Comparisons between groups were achieved using nonpara- levels (Supplemental Figures 4 and 5). Multivariate mod- ϩ Ϫ metric analysis, namely two-tailed Wilcoxon rank sum tests and eling by OPLS discriminated EM Sev from EM , and Kruskal-Wallis tests with Dunn’s post hoc analysis, unless oth- OPLS-derived regression coefficients ranked medium- to erwise stated. Friedman’s analysis was used for paired analysis. very-long-chain GlcCers and SMs (C16:0–C24:1) as major The P values were tested for multiple hypothesis testing using influencing independent variables (Figure 1C and Supple- Bonferroni adjustment or false discovery rate estimation. Lipids with adjusted P Յ .01 or q value Յ0.15 and fold change Ն1.15 mental Figure 5B). Supplemental Figure 5C shows a ro- were deemed significant. The R statistical software environment bust exchange of sphingolipids between the 2 biofluids (http://www.r-project.org/) was used to perform false discovery (serum and PF, Spearman r ϭ 0.9, P Ͻ .0001). doi: 10.1210/jc.2014-1340 jcem.endojournals.org E1917 Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021

Figure 1. Serum sphingolipid profile of endometriosis. A, Heat map representation of median-centered, SD-scaled data from women of Ϫ ϭ ϩ ϭ ϩ ϭ differential endometriotic stages as determined by rAFS classification (EM ,n 24; EM Mild,n 11; EM Sev,n 27). Each row represents a sphingolipid species (total 124 species), whereas each column represents a subject grouped together according to their endometriotic status. B, Ϫ ϩ ϩ Z-score plots of serum sphingolipids in subjects without endometriosis (EM ), mild endometriosis (EM Mild), severe endometriosis (EM Sev), and all ϩ grades of endometriosis (EM All). C, Regression coefficients of te top 10 ranked important variables (sphingolipids) from serum that have potential influence on endometriosis.

Serum and PF GlcCers are increased in (Supplemental Figure 6, A–D). Overall, there were few ϩ Ϫ endometriosis major sphingolipid changes in EM Mild relative to EM . The strong GlcCer correlation with endometriosis led us to explore this because GlcCer, a Role of endometrium in regulating altered mitogenic factor, may be a prime candidate for lesion sub- sphingolipid flux sistence ectopically. In our broad mass spectrometry pro- Because there are numerous molecular defects in the filing of sphingolipids, systematic analysis revealed signif- endometrium that reflect enhanced survival, evasion of icantly higher levels of total serological and PF GlcCer apoptosis, and lesion establishment in endometriosis (27), ϭ ϩ (P .0004 and .005, respectively) in EM Sev patients we determined whether endometrial sphingolipid en- compared with controls (Figure 2). Further examination zymes were altered. At the transcript level, we found up- ϩ ϭ revealed an enrichment of GlcCer and a striking enrich- regulation of SMPD3 (Smpd3)inEM Sev women (n 6) Ϫ ment of very-long-chain monounsaturated sphingolipids relative to EM (n ϭ 6; P ϭ .026; Figure 3A) but not ϩ (SM, Cer, and GlcCer) of 24 carbons (C24:1)inEM Sev serine-palmitoyl transferases (Sptl1–3), suggesting SM hy- women (Table 2), suggesting sequential sphingolipid me- drolysis as the main source of Cer and not through de novo tabolism. Other sphingolipids such as C1P, Cer, and Lac- synthesis. SM can be synthesized by the transfer of phos- ϩ Cer were not significantly enriched in EM Sev women phocholine from PC onto Cers, catalyzed by SMS1e E1918 Lee et al Dysregulated Metabolic Processing in Endometriosis J Clin Endocrinol Metab, October 2014, 99(10):E1913–E1921

Table 2. Fold Change of Serum, PF, and Endometrial Tissue Sphingolipidsa

؉ ؊ Fold Change (EM Sev /EM )

Sphingolipid Serum PF Tissue SM18.1.24.0 1.77 1.29 0.69 SM18.1.22:1 1.12 1.07 0.66 SM18.1.22.0 1.17 1.14 0.81 SM18.1.20:1 1.14 1.13 0.62 SM18.1.20.0 1.18 1.08 0.78 SM18.1.18:1 1.07 1.13 0.76 SM18.1.18:0 1.05 1.07 0.79 Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 SM18.1.16.1 1.12 1.06 0.68 SM18.1.16.0 1.11 1.03 0.73 Cer d18:1/24:1 1.20 1.41 0.36 Cer d18:1/24:0 1.27 1.41 0.37 Cer d18:1/22:1 1.32 1.16 0.49 Cer d18:1/22:0 1.39 1.49 0.36 Cer d18:1/20:0 1.17 1.62 0.49 Cer d18:1/18:0 0.97 1.41 0.47 Cer d18:1/16:1 1.04 0.98 0.64 Cer d18:1/16:0 1.27 1.33 0.40 Cer d18:0/24:1 1.08 1.09 0.65 Cer d18:0/24:0 0.81 1.32 0.31 Cer d18:0/18:1 0.95 1.03 0.34 Cer d18:0/16:0 1.10 1.45 0.35 GluCer d18:1/24:1 1.47 1.14 1.76 GluCer d18:1/24:0 1.47 1.22 0.40 GluCer d18:1/22:0 1.42 1.23 0.53 GluCer d18:1/20:0 1.32 1.37 0.55 GluCer d18:1/18:0 1.47 1.32 0.58 GluCer d18:1/16:0 1.28 1.36 0.32 Figure 2. Accumulation of GlcCer in endometriosis. Scatter plots of GluCer d18:0/24:1 1.21 1.06 2.02 total serum and PF GlcCer in EMϩ and EMϪ subjects. Open boxes, GluCer d18:0/16:0 1.25 1.16 0.57 Ϫ ϭ ϩ ϭ ϩ ϭ LacCer d18:1/22:0 1.35 1.47 0.47 EM (n 24); pink boxes, EM Mild (n 11); red boxes, EM Sev (n ϩ ϭ ϩ LacCer d18:1/16:1 1.20 1.51 0.79 27); maroon boxes, EM All (n 38). Although 2 EM Sev patients seemingly contribute to the difference (blue arrows), their removal LacCer d18:1/16:0 1.24 1.41 0.49 maintained statistical significance (P ϭ .007). P values adjusted by C1P d18:1/24:1 1.35 1.35 0.27 Bonferroni adjustment. C1P d18:1/18:1 1.23 1.14 0.60 C1P d18:1/18:0 1.11 1.46 0.60 C1P d18:1/16:0 1.32 1.20 0.49 C1P d18:0/16:0 1.32 1.17 0.46 (Sgms1). In ovarian endometriosis, several plasma and PF a PCs were found elevated (28, 29). Consistent with this, Only sphingolipids that are statistically significant in at least 1 biological compartment are shown. there was Sgms1 upregulation (P ϭ .041), corresponding decreases in tissue PCs, and increases in serum and PF PCs ϩ Ϫ ϭ ϭ ϭ Ϫ ϩ in EM Sev relative to EM (P .038, P .0009, P .039 similar between EM and EM Sev (Supplemental Figure respectively; Supplemental Figure 6E). Importantly, UDP- 7). GalCer synthase expression levels were similar be- Cer glucosyltransferase (Ugcg; GCS) levels were tween EMϪ and EMϩ subjects, further supporting our ϩ ϭ higher in EM Sev subjects (P .041). Upregulation of LC-MS/MS measurements of GlcCer elevation, and not Ugcg transcript in endometriotic lesions of a rat model of GalCer in endometriosis (Supplemental Figure 7). Endo- endometriosis has been previously reported (30). Our data metriotic endometrial cells are more proliferative (31), thus indicated the flux of EMϩ endometrial sphingolipid and given the upregulation of endometrial GCS, elevated 3 3 3 ϩ ϩ metabolism: PC SM Cer GlcCer (Figure 3B). By GlcCer should be found in EM Sev endometrium. EM Sev/ IHC, increased SMS1, SMPD3, and GCS expression were EMϪ fold change of endometrial very-long-chain GlcCer ϩ ϭ detected in EM Sev glandular epithelial and stromal cells (C24:1) were high (n 7 per group; Table 2); by contrast, (n ϭ 6), whereas these enzymes were expressed predom- most sphingolipids were elevated in serum and PF but low inantly in the glands in EMϪ specimens (n ϭ 6; Figure 3C in endometrium, suggesting endometrium as the tissue of and Supplemental Figure 7). In concordance with our origin (Table 2 and Figure 4, A–C). There was a corre- ϩ quantitative RT-PCR results, staining intensities for sponding decreased apoptosis in EM Sev endometrial tis- SMPD1, SMPD2, and serine-palmitoyl transferase were sues (Figure 4D) relative to EMϪ women, consistent with doi: 10.1210/jc.2014-1340 jcem.endojournals.org E1919 Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021

Figure 3. Regulation of sphingolipid concentrations in endometriosis in the endometrium. A, Upregulation of endometrial sphingolipid enzymes ϩ ϭ Ͻ in EM Sev (n 5 per group). *, P .05. B, Serum lipid concentrations and lipid enzymes that control their metabolism in the endometrium of ϩ Ϫ EM Sev and EM . Colors indicate fold changes in the expression levels of lipid enzymes that regulate the flux of sphingolipids. Font sizes are proportionate to the abundance of the lipids in serum. Unmeasured analytes are shown in gray. C, Endometrial sphingolipid enzyme expression levels as measured by fluorescence intensity in stromal (blue bar) and epithelial (red bar) compartments (n ϭ 6). *, P Ͻ .05 for stromal comparison; and #, P Ͻ .05; ##, P Ͻ .01 for epithelial comparisons. previous reports (32, 33), an observation in line with the systems biology that aims to investigate and model lipids crucial role of GlcCer as a proliferative agent. Overall, at the global level. This is done in part in an effort to stitch these findings suggest that the accumulation of GlcCer in lipids with the rest of the ’omics’ sciences such as pro- endometriosis is due to aberrant sphingolipid metabolism teomics and genomics to comprehensively understand bi- in the endometrium that may render eutopic endometrial ological systems at the systems level. In this study, we used cells more proliferative. targeted LC-MS/MS-based lipidomics in a case-control study for the profiling of Ͼ120 sphingolipids to evaluate sphingolipid changes in endometriosis systematically. Discussion This sphingolipidome-wide quantitative analysis identi- Sphingolipids define a distinctive and important lipid class fied the in vivo accumulation of GlcCer in endometriotic involved in diverse biological functions including signal women as a result of altered sphingolipid metabolic pro- transduction and cell fate determination, but their asso- cessing by GCS in the endometrium. ciation with endometriosis pathophysiology remains The association of GlcCer accumulation and hyperpro- poorly studied. Lipidomics is an emerging discipline in liferation and organomegaly seen in human and experi- E1920 Lee et al Dysregulated Metabolic Processing in Endometriosis J Clin Endocrinol Metab, October 2014, 99(10):E1913–E1921

proliferation and potentially the growth of endometriotic lesions. Re- cent studies have also shown that sphingosine-1-phosphate receptors are upregulated in EMϩ ectopic and eutopic endometrium (38), and it re- sults in increased viability upon ex- posure to sphingosine analogs (37), further suggesting the role of dys- regulated sphingolipid metabolism in this disease. Together, our data Downloaded from https://academic.oup.com/jcem/article/99/10/E1913/2836173 by guest on 25 September 2021 shed some light on the importance of dysregulated sphingolipid fluxes and the specific effects of accumulated GlcCer in endometriosis, suggesting a possible pathophysiological role. Whatever the etiological agent of the observed accumulation of GlcCer is, approaches that target this pathway may address both the establishment and sustenance of endometriotic le- sions concurrently. Experimental models will be helpful in clarifying the plausible biological roles of these sphingolipids in the development of enhanced cell proliferation and le- sion development. It should be rec- Figure 4. Accumulation of GlcCer in endometriosis. A, Scatterplot of mean serum sphingolipid levels ognized that our study is exploratory against mean endometrial tissue sphingolipid levels. B, Scatter plot of mean PF sphingolipid levels against mean endometrial tissue sphingolipid levels. GlcCers are highlighted in red. C, Because the tissue and PF/ and requires further studies in an in- serum data were not necessarily from the same patients, Fisher’s exact test and Wilcoxon P values of dependent population cohort for demographics variables in EMϩ patients were tested and shown to be similar. D, Apoptotic index based Sev both biomarker discovery and vali- on average number of apoptotic bodies/6.4 mm2 detected by TUNEL IHC of EMϩ and EMϪ women (n ϭ 4–5). *, P Ͻ .05. Original magnification, ϫ400; scale bar, 50 ␮m. dation purposes. In conclusion, our clinical sphin- golipidomics approach defines the mental diseases such as Gaucher’s disease, polycystic kid- net outcome of an imbalanced GlcCer that may be crucial ney disease, and diabetes demonstrate the effect of for endometriosis pathophysiology. Such heightened pro- dysregulated GlcCer metabolic processing on tissue pro- liferative abilities may be key to how menstrual fragments liferation (15, 34). In animal models and cellular work, may persist as endometriotic lesions in ectopic sites. GlcCers possess growth-stimulatory effects, and pharma- cological or genetic inhibition of GCS resulted in cell death (13, 16, 35, 36). Consistent with this, we identified in- creased endometrial GCS expression and corresponding Acknowledgments serum and PF GlcCer accumulation in women with endo- We acknowledge the help of Drs. Heng Hao Tan, Mathew Lau metriosis, raising the possibility of biochemically induced and Steven Teo from KK Women’s and Children’s Hospital in the proliferative abnormalities in the endometrium. It is there- collection of samples. fore interesting that we found increased serum and PF Cer in EMϩ women that would have promoted apoptosis of Address all correspondence and requests for reprints to: Jerry shed endometrial cells. Endometrial stromal cells origi- K. Y. Chan, MD, PhD, Reproductive Medicine, KK Women’s nating from EMϩ women have blunted responses to the and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899. E-mail: [email protected]. apoptosis-inducing effects of Cer (32, 37). Consequently, This work was supported by the Singapore National Research the combination of GlcCer to promote cell proliferation Foundation (to Singapore-MIT Alliance for Research and Tech- and apoptotic-resistant endometriotic cells suggest intrin- nology). J.K.Y.C. received salary support from the National sic cellular dysregulation conducive for the enhanced cell Medical Research Council (CSA/043/2012). doi: 10.1210/jc.2014-1340 jcem.endojournals.org E1921

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