Original Article

Elevated Systemic Levels of Endocannabinoids and Related Mediators Across the Menstrual Cycle in Women With Endometriosis

Reproductive Sciences 1-9 ª The Author(s) 2016 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719116630414 rs.sagepub.com

Ana Maria Sanchez, PhD1, Raffaella Cioffi, MD2, Paola Vigano`, PhD2, Massimo Candiani, MD2, Roberta Verde, BSc3, Fabiana Piscitelli, PhD3, Vincenzo Di Marzo, PhD3, Elisabetta Garavaglia, MD2, and Paola Panina-Bordignon, PhD1

Abstract Cannabinoids and modulators of the endocannabinoid system affect specific mechanisms that are critical to the establishment and development of endometriosis. The aim of this study was to measure the systemic levels of endocannabinoids and related mediators in women with and without endometriosis and to investigate whether such levels correlated with endometriosisassociated pain. Plasma and endometrial biopsies were obtained from women with a laparoscopic diagnosis of endometriosis (n ¼ 27) and no endometrial pathology (n ¼ 29). Plasma levels of endocannabinoids (N-arachidonoylethanolamine [AEA] and 2-arachidonoylglycerol [2-AG]) and related mediators (N-oleoylethanolamine [OEA] and N-palmitoylethanolamine [PEA]), messenger RNA expression of some of their receptors (cannabinoid receptor type 1 [CB1], CB2, transient receptor potential vanilloid type [TRPV1]), and the enzymes involved in the synthesis (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D [NAPE-PLD]) and degradation (fatty acid amide hydrolase 1 [FAAH]) of AEA, OEA, and PEA were evaluated in endometrial stromal cells. The systemic levels of AEA, 2-AG, and OEA were elevated in endometriosis in the secretory phase compared to controls. The expression of CB1 was higher in secretory phase endometrial stromal cells of controls versus endometriosis. Similar expression levels of CB2, TRPV1, NAPE-PLD, and FAAH were detected in controls and endometriosis. Patients with moderateto-severe dysmenorrhea and dyspareunia showed higher AEA and PEA levels than those with low-to-moderate pain symptoms, respectively. The association of increased circulating AEA and 2-AG with decreased local CB1 expression in endometriosis suggests a negative feedback loop regulation, which may impair the capability of these mediators to control pain. These preliminary data suggest that the pharmacological manipulation of the action or levels of these mediators may offer an alternative option for the management of endometriosis-associated pain. Keywords endometriosis, endocannabinoids, pain

Introduction Endometriosis is a benign, chronic, inflammatory disease that affects 10% of reproductive-age women and up to 50% of women with infertility.1 Symptoms include dysmenorrhea, dyspareunia, chronic pelvic pain, irregular uterine bleeding, and/or infertility. The search for novel therapeutics is a challenging issue, aimed at identifying a drug that prevents recurrences, allows pain management, and continued attempts to conceive.2 Among the various candidates, endocannabinoids and modulators of the endocannabinoid system have recently emerged as potential pharmacological targets in endometriosis, as they affect specific mechanisms that are critical to disease establishment and maintenance.3-10 Endocannabinoids play an

1 Division of Genetics and Cell Biology, Reproductive Sciences Laboratory, IRCCS Ospedale San Raffaele, Milan, Italy 2 Department of Obstetrics and Gynecology, IRCCS Ospedale San Raffaele, Milan, Italy 3 Endocannabinoid Research Group, CNR, Pozzuoli, Italy

Corresponding Author: Paola Panina-Bordignon, Division of Genetics and Cell Biology, Reproductive Sciences Laboratory, IRCCS Ospedale San Raffaele, Via Olgettina 58, 20132 Milan, Italy. Email: [email protected]

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Reproductive Sciences

Table 1. Clinical Characteristics of Enrolled Patients: Distribution, Progesterone Levels (P), Age, and Diagnosis. Groups (n)



Age, Mean + SD

Cases (n ¼ 27)

n ¼ 20

n ¼ 14

36.60 + 1.19

Controls (n ¼ 29)

n ¼ 20

n ¼ 12

36.81 + 1.40

Diagnosis, n (%) Peritoneal endometriosis (13/27 ¼ 48.1%) Ovarian endometrioma (14/27 ¼ 51.8%) Bladder nodules (2/27 ¼ 7.4%) Scar endometrioma (1/27 ¼ 3.7%) Deep endometriosis (3/27 ¼ 11.1%) Sactosalpinx (13/29 ¼ 44.8%) Pelvic inflammatory disease (1/29 ¼ 3.4%) Asherman syndrome (1/29 ¼ 3.4%) Ovarian mature teratoma (6/29 ¼ 20.6%) Serous cystadenoma (3/29 ¼ 10.3%) Bartholin gland cyst (3/29 ¼ 10.3%) Anterior prolapse (1/29 ¼ 3.4% Idiopathic infertility (1/29 ¼ 3.4%)

Abbreviation: SD, standard deviation.

important role in several female reproductive processes, including folliculogenesis, ovulation, and oocyte maturation as well as implantation and early pregnancy.11 In addition, exogenous cannabinoids have been shown to decrease endometriosis-associated hyperalgesia in a rat model and in humans.4,6 The endocannabinoid system comprises bioactive lipid mediators, that is, the endocannabinoids; enzymes involved in their synthesis and degradation; and the cannabinoid (CB) receptors, which are activated by endocannabinoids.12 The most comprehensively studied endocannabinoids are the fatty acid amide, N-arachidonoylethanolamine (AEA or anandamide), and 2-arachidonoylglycerol (2-AG).13 Two AEA congeners, Npalmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA), often accompany endocannabinoids in tissues and share biosynthetic and catabolic pathways with AEA, although they activate noncannabinoid receptors (see subsequently). The biosynthesis of AEA, which occurs ‘‘on demand,’’ is mainly catalyzed by N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). Following its synthesis, AEA is transported to the extracellular space to bind specific receptors, including CB1, largely expressed in the central nervous system but also in the liver, heart, adipose tissue, endometrium, and ovary, and CB2, mainly expressed in immune cells and other peripheral tissues. N-arachidonoylethanolamine is then inactivated by the serine hydrolase enzyme fatty acid amide hydrolase 1 (FAAH). Unlike AEA (and 2-AG), PEA and OEA have no direct agonist activity at cannabinoid CB1 and CB2 receptors and preferentially bind to peroxisome proliferator-activated receptor a. In addition, AEA, 2-AG, OEA, and PEA also activate the transient receptor potential vanilloid type 1 (TRPV1), a nonselective cation channel involved in nociception in the periphery and in the central nervous system.14,15 Many components of the endocannabinoid system, CB1 and CB2 receptors, and the enzymes NAPE-PLD and FAAH coexist in many cell types in the endometrium.16 The expression and function of TRPV1 have been demonstrated in ectopic endometrial stromal cells (ESCs) when stimulated with inflammatory mediators.17 It has been recently demonstrated that the anti-inflammatory mediator PEA

enhances the levels of 2-AG and potentiates its activation and desensitization of TRPV1 channels.18 N-arachidonoylethanolamine and 2-AG are expressed in reproductive tissues, and their role in reproductive function is well established.19 The uterus contains the highest concentration of AEA20 and the main components of the endocannabinoid system in variable concentration across the menstrual cycle.16 The expression of CB1 receptors is significantly lower in the eutopic endometrium of women with endometriosis compared with controls, independent of the cycle phase.21 N-arachidonoylethanolamine and 2-AG are also produced by immune cells in response to inflammation,22 and these modulate proinflammatory mediator release and cell migration in early pregnancy.23 Cells in inflamed tissues generate a variety of proalgesic lipid mediators, which increase the excitability of nociceptive neurons. The proalgesic influence of these mediators is balanced by the actions of AEA, 2-AG, and PEA, which inhibit pain initiation by regulating the transmission of nociceptive signals from peripheral inflamed sites to the central nervous system.24 In the current study, we evaluated the expression of some of the best-characterized components of the endocannabinoid system in the plasma and in ESCs of women with and without endometriosis across the menstrual cycle. Finally, we correlated the systemic levels of these lipid mediators with pain symptoms.

Materials and Methods Patients Endometrial tissues were collected from 56 caucasian women (age, 22-45 years) undergoing laparoscopy for benign gynecological disorders at the Department of Gynecology and Obstetrics of the San Raffaele Hospital (Milan, Italy; Table 1). Endometrial biopsies were obtained through an uterine curettage after hysteroscopy. Laparoscopic examination demonstrated the presence of endometriosis in 27 patients (I-IV rAFS [revised American Fertility Society score] stage; Table 2). The control group was composed of 29 women without laparoscopic evidence of

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Sanchez et al


Table 2. Clinical Characteristics of Enrolled Patients: rAFS Stage and VAS Score. Groups

rAFS, Mean + SD

rAFS Stage, n/Total, %

Cases (n ¼ 27)

38.9 + 26.42

I: 2/27, 7.4% II: 4/27, 14.8% III: 13/27,48.1% IV: 8/27, 29.6% NA

Controls (n ¼ 29)


Infertility, n/Total (%)

AMH ng/mL, Mean + SD

Dysmenorrhea: 65.63 + 37.77 Dyspareunia: 23.57 + 33.88 Chronic pelvic pain: 29.29 + 29.21

12/27 (44.4%)

1.809 + 0.40 (n ¼ 18)


11/29 (37.9%)

2.215 + 0.77 (n ¼ 10)

VAS Score, Mean + SD

Abbreviations: NA, not available; SD, standard deviation; VAS, visual analog scale; rAFS, revised American Fertility Society Score; AMH, anti-mullerian hormone.

endometriosis (Table 1). Exclusion criteria for all donors included cancer, recent (

Elevated Systemic Levels of Endocannabinoids and Related Mediators Across the Menstrual Cycle in Women With Endometriosis.

Cannabinoids and modulators of the endocannabinoid system affect specific mechanisms that are critical to the establishment and development of endomet...
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