{"paper_id":"77c1cc4b-a484-4ace-8401-ea1cc0d70d98","body_text":"The study was approved by the Lothian Research Ethics Committee (LREC 11/AL/0376). Peritoneal biopsies [∼1.0 × 0.5 cm size: stored in RNAlater (Applied Biosystems) at −20°C] were obtained from 3 groups of women (aged 18–45 y) at the time of surgery with informed consent: 1) women undergoing laparoscopic investigation for CPP with histological evidence of pelvic endometriosis (n = 12); 2) women undergoing laparoscopic investigation for CPP without evidence of endometriosis (n = 10); and 3) women undergoing laparoscopic sterilization without CPP and without evidence of endometriosis (n = 5). Endometriosis lesions were also collected from women with endometriosis (n = 18). Peritoneal biopsies from women without endometriosis were recovered from a site prone to endometriosis (pouch of Douglas) and in women with endometriosis the peritoneal biopsy was recovered from a site adjacent to the endometriosis lesion. None of the women had taken exogenous hormones for at least 3 months at the time of sampling.\nHuman ES cells, strain H9 (WiCell) were cultured on inactivated mouse embryonic fibroblasts maintained in Knockout DMEM/F12 supplemented with 20% knockout serum replacement (Gibco), 1 mM L-glutamine, 100 μM MEM nonessential amino acids, and 0.1 μM β-mercaptoethanol, with 6 ng/mL FGF-2 (R&D Systems). Differentiation of ES cells was induced using a cocktail of small molecule inhibitors as detailed by Chambers et al ( 15 ). RNA was extracted from cells at selected time points for the characterization and verification of phenotype. On day 22 the sensory neurons were trypsinized and reseeded into 12-well plates at equal densities (5 × 10 −5  cells/well). On day 25, the cells were treated for 24 hours with 10 −8  M 17β-estradiol (E2; Sigma), the estrogen receptor (ER)-α selective agonist 4,4′,4′-[4-propyl-(1H)-pyrazole-1,3,5-tryl] trisphenol (PPT), or the ERβ selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN; Tocris) alone or in combination with the antiestrogen fulvestrant (ICI; 10 −7  M; Tocris) dissolved in dimethylsulfoxide (DMSO).\nExpression of neurofilament protein was detected using neurofilament H chicken antineurofilament H (1:1000; Covance); nuclei were stained with 4′,6′-diamino-2-phenylindole and images captured on an Axiovert microscope (Carl Zeiss Inc).\nNeurons were stimulated with 4 nM capsaicin (Sigma), and intracellular calcium was measured using a calcium indicator kit (BD Biosciences) with calcium flux captured using a NOVOstar microplate fluorometer (BMG Labtech).\nRNA was extracted from human tissues by homogenization in TRI reagent, chloroform phase separation and the lysates processed using an RNAeasy kit (QIAGEN). RNA was extracted from cells using RLT (lysis) buffer and an RNAeasy kit. Samples were deoxyribonuclease treated (QIAGEN) and concentration and purity assessed using a NanoDrop ND 1000. cDNA was synthesized using SuperScript VILO enzyme (Invitrogen) with 100 ng starting template in a 20-μL reaction. PCRs (15 μL) were performed using the Roche Universal Probe Library (Roche Applied Science) and Express quantitative PCR supermix (Invitrogen) with primers added at a concentration of 20 μM and thermal cycling performed on a 7900 Fast real-time PCR machine (Applied Biosystems) with  18S  selected as the reference gene because we have confirmed this gene is not altered by estrogenic treatments. cDNA was added at 1.5 μL per reaction and duplicate technical replicates performed.\nData were analyzed with RQ manager software (Applied Biosystems) using the δδCt method, and samples were normalized to either one laparoscopic sterilization sample (patients) or one vehicle control sample (cells). Primer sequences included the following:  TRPA1 , forward, 5′-tggacaccttcttcttgcatt-3′, reverse, 5′-tcatccatttcatgcagcac-3′;  TRPV1 , forward, 5′-agagtcacgctggcaacc-3′, reverse, 5′-ggcagagactctccatcacac-3′;  SCN9A , forward, 5′-caacttttaagggatggacga-3′, reverse, 5′-tcatatttgggctgcttgtct-3′;  SCN11A , forward, 5′-acctgagcctgaacaacagg-3′, reverse, 5′-tttgaactctctggctcgtg-3′;  P2RX3 , forward, 5′-ggcctttacttctgtgggagt-3′, reverse, 5′-aaacttcttggctttgtactggtc-3′;  TAC1 , forward, 5′-gcctcagcagttctttggat-3′, reverse, 5′-agcctttaacagggccactt-3′;  ER α, forward, 5′-ttactgaccaacctggcaga-3′, reverse, 5′-atcatggagggtcaaatcca-3′; and  ER β, forward, 5′-atcatggagggtcaaatcca-3′, reverse, 5′-tgggcattcagcatctcc-3′.\nQuantitative PCR (QPCR) data were analyzed using a one-way ANOVA and a Newman Keuls post hoc multiple comparison test. Statistics were generated using GraphPad Prism 6 software.\n\nMeasurement of mRNAs encoded by  TAC1  and nociceptive ion channels revealed differences between tissue samples;  TAC1  was elevated only in endometriosis lesions ( Figure 1 A;  P  < .05), and  P2RX3  was increased in the peritoneum of women with CPP, regardless of whether they were diagnosed with endometriosis compared with the peritoneum of healthy women ( Figure 1 B;  P  < .05).  SCN9A  (Nav1.7) was elevated in lesions from women with endometriosis ( Figure 1 C;  P  < .05), whereas  SCN11A  was significantly higher in the peritoneum of women with CPP and endometriosis compared with the peritoneum of women with CPP alone ( Figure 1 D;  P  < .001). mRNA encoded by  TRPA1  was significantly increased in the peritoneum of women with endometriosis compared with the peritoneum of healthy women and those with CPP alone ( Figure 1 E;  P  < .001).  TRPV1  was elevated in peritoneum ( P  < .01) and in lesions ( P  < .05) of women with endometriosis compared with the peritoneum of healthy women ( Figure 1 F).\nThe neuropeptide  TAC1  and nociceptive ion channels are differentially expressed in CPP and in endometriosis. A–F, mRNA concentrations of the neuropeptide  TAC1  (A) and the nociceptive ion channels  P2RX3  (B),  SCN9A  (C),  SCN11A  (D),  TRPA1  (E), and  TRPV1  (F) were analyzed using QPCR. Concentrations of mRNAs were measured in the peritoneum of women with no pain (control; n = 5), compared with the peritoneum of women with CPP but no obvious underlying pathology (PP; n = 10), and the peritoneum (EP; n = 12) and peritoneal lesions (EL; n = 18) of women with confirmed endometriosis. RQ, relative quantification. Statistical analysis was performed using a one-way ANOVA combined with a Newman Keuls post hoc test. *,  P  < .05; **,  P  < .01; ***,  P  < .001. Asterisks denote statistical significance compared with control, and lines and asterisks denote significance between specific groups.\nSensory neurons immunopositive for neurofilament were generated in vitro ( 15 ) ( Figure 2 , A–C). Differentiation was accompanied by a reduction in octamer transcription factor-4 mRNA concentrations and an increase in  TAC1, SCN9A , and  SCN11A  ( Figure 2 D), consistent with differentiation of sensory neurons with a nociceptor-like phenotype. When the differentiated cells were incubated with capsaicin (activates TRPV1), an intracellular calcium flux was detected ( Figure 2 E). The expression of ER subtypes changed during differentiation;  ER α mRNA concentrations decreased and  ER β mRNAs increased ( Figure 2 F). Stimulation of sensory neurons with the estrogen ligands E2, DPN, and PPT revealed ER-dependent regulation of nociceptive ion channels.  TAC1  and  P2RX3  mRNAs were increased after the incubation with DPN compared with cells incubated with DPN in the presence of ICI ( Figure 2 G;  P  < .001) or vehicle control (DMSO;  Figure 2 H;  P  < .05), respectively.  TAC1  was also elevated by PPT compared with PPT + ICI ( Figure 2 G;  P  < .01). Although  SCN9A  increased with DPN ( P  < .05), this was not blocked by the addition of ICI ( Figure 2 I), and the expression of  SCN11A  ( Figure 2 J) and  TRPA1  ( Figure 2 K) did not appear to be ER dependent. In contrast,  TRPV1  mRNA concentrations were elevated by both E2 ( P  < .05) and DPN ( Figure 2 I;  P  < .01), and this effect was abrogated by ICI.\nmRNA concentrations of nociceptive markers are regulated by estrogens in an in vitro model of human sensory neurons with a nociceptor-like phenotype derived from ES cells. A, Human ES cells were differentiated into sensory neurons with a nociceptor-like phenotype using combined small molecule inhibitors. Sensory neurons developed projections (B) and stained positively for neurofilament using immunocytochemistry (C). Scale bar, 200 μM. D, During the differentiation method, RNA was extracted from cells at different time points: pluripotent stem cells (d −7, n = 3), day 1 (n = 4), day 3 (n = 4), day 5 (n = 6), day 7 (n = 4), day 9 (n = 3), day 11 (n = 3), day 17 (n = 3), and day 21 (n = 3). QPCR analysis revealed that mRNA concentrations of the pluripotency marker octamer transcription factor-4 ( Oct4 ) declined as the differentiation procedure progressed. The nociceptive markers  TAC1, SCN9A , and  SCN11A  increased. E, Sensory neuron functionality was determined by stimulating cells with 4 nM capsaicin and recording calcium flux. F, During differentiation from ES cells to mature sensory neurons,  ER α mRNAs declined, whereas  ER β inclined. G–L, Mature sensory neurons were incubated with DMSO (vehicle), E2, DPN, or PPT, with or without ICI, and RNA was extracted after 24h. G–I, Using QPCR  TAC1  mRNAs were elevated by DPN and PPT compared to DPN + ICI and PPT + ICI.  P2RX3  and  SCN9A  mRNAs were elevated by DPN compared with vehicle control (DMSO). J and K, No significant differences were detected in  SCN11A  and  TRPA1 . L,  TRPV1  mRNAs were elevated by E2 and DPN. RQ, relative quantification. Statistical analysis was performed using a one-way ANOVA and Newman Keuls post hoc test. *,  P  < .05; **,  P  < .01; ***,  P  < .001. Asterisks denote statistical significance compared with vehicle control (DMSO), and lines and asterisks denote significance between specific treatment groups.\n\nWe believe this is the first study to detect differences in the expression of mRNAs encoded by the ion channels  TRPA1 ,  P2RX3 ,  SCN9A , and  SCN11A  in the peritoneum and lesions of women suffering from CPP, some of whom had endometriosis. We believe these insights may be indicative of sensitization of nerves present within the peritoneal lining of women with pain and therefore shed new light on the mechanisms responsible for development of chronic hypersensitivity.\nIn women with active endometriosis, we discovered that the concentrations of mRNAs encoded by  SCN11A ,  TRPA1 , and  TRPV1  were all significantly increased in the samples of peritoneum. We believe these novel findings are consistent with nociceptive changes taking place within the peritoneum of women with endometriosis, suggesting an increased sensitization of the peritoneum adjacent to the lesion. We detected significant increases in  TRPV1  mRNAs in both the peritoneum and in lesions, confirming and extending a previous study ( 11 ). Notably,  P2RX3  mRNAs were significantly elevated in the peritoneum of women with CPP, regardless of whether they had active endometriosis and would be consistent with a neuropathic component to idiopathic CPP.\nTo date, researchers have used rat or chick dorsal root ganglia (DRG) neurons as an in vitro model to investigate the function of sensory neurons. However, because DRG neurons represent a mixed population of somatosensory neurons, the development of a differentiation protocol allowing the generation of a population of human sensory neurons with a nociceptor-like phenotype ( 15 ) is particularly welcome. In the current study, we detected expression of estrogen receptor mRNAs in our ES cell-derived cultures. Treatment of cells with E2 or the ERβ agonist DPN induced a significant increase in  TRPV1  mRNAs that was blocked by the addition of the ER antagonist ICI. In addition,  P2RX3, SCN9A , and  TAC1  mRNAs were all increased when cells were incubated with the ERβ agonist DPN, and  TAC1  was also increased by PPT. We have previously demonstrated DPN-dependent changes in gene expression in endometrial endothelial cells and have shown that these were mediated via specificity protein-1 tethered transcription ( 16 ). Bioinformatics revealed the presence of specificity protein-1 transcription factor binding sites in the promoter regions of  TRPV1 ,  P2RX3 ,  SCN9A , and  TAC1  (Match; Biobase international.com).  TRPV1  mRNA was the only transcript that was increased in response to E2 treatment and abrogated by the antiestrogen ICI. Notably, bioinformatics detected several estrogen response elements in the promoter region of  TRPV1  and throughout the length of the transcript (Dragon estrogen response element locator version 6.0; National Institute of Health) that could facilitate direct binding of E2-activated ER receptor dimers. Further studies are required to validate these findings.\nEstrogens are reported to have both pro- and antinociceptive properties. Mice exhibit hyperalgesia when estrogens are depleted after ovariectomy ( 17 ), and nociceptive responses are lower in ERβ knockout mice during the early stages of inflammation ( 18 ). The results of our study suggest estrogens may have a direct impact on nociception by up-regulating the expression of ion channels and would be consistent with reports that the expression of Trpv1 and P2x3 proteins are decreased in DRGs from mice with targeted deletions of  Esr2  and  Esr2  ( 19 ).\nIn summary, the results of this study indicate that therapies targeting P2RX3 may be useful in treating CPP in women with a diverse range of painful etiologies, whereas women with active endometriosis could benefit from treatments targeting TRPV1, TRPA1, SCN9A, or SCN11A. Studies using sensory neurons suggest regulation of TRPV1 by estrogens may provide an explanation for the reduction in pain experienced when ovarian steroids are suppressed and an opportunity for development of novel therapies.","source_license":"CC0","license_restricted":false}