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1 Siena, Piazza del Campo : 0 0: 770 years yeas of history stoy Siena, Policlinico Universitario Santa Maria alle Scotte
2 Endometriosis: symptoms Infertility Pain
3 Endometriosis and infertility: possible mechanisms Follicular environment and ovulation Changes in peritoneal fluid Adhesions between peritoneum and pelvic organs Eutopic endometrium abnormalities Implantation disorders Endometriosis, 2 edition. Oxford, UK: Health Press 2003
4 Hormones and inflammation:endometriosis Estrogens Progesterone Cytokines Chemokines Free radicals (Oxidative stress) Prostaglandins + - Endometrial cell Cell proliferation and differentiation Immune function and inflammation Apoptosis Invasion / neoangiogenesis Chronic Inflammation Endometriotic cell Ge enetic/ /epigen epigen netic i influen nces
5 Endometriosis: i tissues studied d Fallopian tube Ovary (endometrioma) Peritoneal lesions Eutopic endometrium Ectopic endometrium But also: - Gastrointestinal lesions - Urinary tract
6 Eutopic and ectopic endometrium: hormone receptors, factors and enzymes ER Angiogenetic g factors Glycodelin PRA/B Oxidative stress markers TGF- related factors (inhibin and activin) Cytokines Aromatase Metalloproteinases Growth factors Neuropeptides
7 Neuroendocrine factors in endometriosis In women with endometriosis, there is abnormal sprouting of nociceptors in the endometrium and in peritoneal endometriotic lesions Nerve fibres Jabbour et al. SGI 2010
8 Neuroendocrine factors in endometriosis Substance P VIP Neuroendocrine cells Synaptophysin (SYN) Neuropeptide Y Calcitonine gene related protein Bokor et al, 2009 and Wang et al., 2010 neuron-specific enolase (NSE)
9 Neuroendocrine system is localized in various organs and tissues Neurohormones Neuropeptides p Neurotransmitters Adrenal glands Gastrontestinal tract Neuroendocrine Reproductive organs tract Russo et al, Acta Histoch 2006
10 Endometrium and neuroendocrine factors Prolactin CRH Endometrium Urocortins Paracrine action Relaxin Substance P Vasoactive Intestinal peptide Autocrine action
11 CRH is a neuropeptide originally localized in central nervous system Stress Hypotalamus CRH Pit itar Pituitary Adrenal ACTH secretion Cortisol secretion
12 CRH-family CRH amino acids 41 %CRH sequence homology urocortin Vaughan et al., Nature 1995 urocortin 2 (orstresscopin - related peptide) Reyes et al., PNAS U S A urocortin 3 (or stresscopin) Lewis et al., PNASUSA S A. 2001
13 CRH- Receptors affinity CRH CRH-R1 +++ Ucn CRH-R2 Ucn Ucn 3
14 Human endometrium express and localize CRH Urocortin Urocortin 2 Urocortin 3 CRH-R1 CRH-R2
15 Endometrial CRH/Ucn and menstrual cycle CRH Ucn CRH mrna expression is higher during secretory phase (Zoumakis et al. 2001) U RNA i i hi h d i t h Ucn mrna expression is higher during secretory phase (Torricelli et al, Hum Reprod 2007)
16 Endometrial cells and CRH receptors Proliferative phase Proliferative phase Secretory phase Secretory phase Endometrial cells express CRH-R2 R2 mrna in both Endometrial cells express phases of menstrual cycle CRH-R1 mrna in both phases of menstrual cycle Karteris et al., JCE&M 2004 Di Blasio et al., JCE&M 1997
17 CRH stimulates decidualization in endometrial stromal cells HESC CRH addition to culture media Decidualized HESC Decidualization Zoumakis et al., Mol Hum Rep 2000 Ferrari et al., J Steroid Biochem Mol Biol.1995
18 Ucn stimulates decidualization in endometrial stromal cells Ucn and prolactin concentration Increase during in vitro decidualization Ucn stimulates decidualization process Torricelli et al., Hum Reprod 2006
19 CRH/CRH-R1 are involved in embryo implantation Mouse model Antalarmin Block of CRH-R1 Makrigiannakis i ki et al., Am J Reprod Immun 2004 Implantation failure70% Kalantaridou et al., Crit Rev in Clinic Lab Sci 2007
20 Endometriosis and CRH CRH pg/ml CRH-BP ng/ml 1,6 25 1,2 20 0,8 15 0, control stage I stage II stage III 5 Florio et al., J Endocrn Invest 1998
21 Endometriosis and Ucn Ectopic tissue A CN1 / HPRT mrna expre ession U Relative Ec topic Ectopic tissue express Ucn mrna
22 Urocortin levels in ovarian endometriosis 250 *# Urocort tin (pg/m ml) * 50 0 Plasma Peritoneal PF Cyst Cystic fluid fluid Obstet Gynecol 2007
23 Endometrium and Ucn Pla asma urocorti in (pg/ml) P< Endometriosis Control Ucn level l detects 88% of cases of endometrioma with a specificity of 90% Florio et al, Obst Gyn 2009
24 Eutopic endometrium in endometriosis: impaired i CRH and Ucn mrna expression ssion CRH /HPRT R elative mr NA expre B ** Control Endometriosis UCN1 / HPRT Relative mrn NA expressio on A A * Control C Proliferativ Proliferative phase Secretory Secretive phase Endometriosis Submitted 2010
25 Eutopic endometrium in endometriosis: role of CRH and Ucn in in vitro decidualization Prola actin (ng/ml) A *** * * d0 d2 d4 d6 d8 Healthy women ***CRH ** * ** Ucn Sham Ast2b Ant sion CRH H-R1/18s Relative mr RNA express A *** * *** d0 d6 d8 healthy endometriosis Prola actin (ng/ml l) B CRH Ucn Ast2b Sham Ant d0 d2 d4 d6 d8 Endometriotic women A CRH-R -R2/18s mrn Relative mrn exp pression NA expression Submitted B d0 d6 d8
26 Role of CRH and Ucn in endometriosis CRH and Ucn showed aberrant cycle-related changes in endometriosis The lack of increase of CRH and Ucn during secretory phase may be involved in a deranged decidualization Endometriotic cells show a deranged expression of CRH-R1 Infertility
27 CRH-family CRH amino acids 41 %CRH sequence homology urocortin Vaughan et al., Nature 1995 urocortin 2 (orstresscopin - related peptide) Reyes et al., PNAS U S A urocortin 3 (or stresscopin) Lewis et al., PNAS U S A. 2001
28 Endometrium and Ucn2/Ucn3 Proliferative phase Secretory phase UCN2 UCN3 Florio et al, Fertil and Steril 2006
29 Endometrium and Ucn2/Ucn3 Ucn2 /1 18s mrna ra atio * 3 ** 2 1 Ucn3/1 18s mrna ratio * ** EP LP ES LS EP LP ES LS Ucn2 Ucn3 Submitted 2010
30 Endometriosis and Ucn2/Ucn3 Ectopic lesions express and localize Ucn2 and Ucn3 mrna UCN2 UCN2 / HP PRT Re elative mrna ex xpression Ectopic UCN3 RT xpression UCN3 / HP elative mrna ex Re * Submitted Ectopic
31 Ucn2 /Ucn3 in endometrium of endometriotic ti women Ucn2/18s mrna ra atio * * Ucn3/18 8s mrna ratio EP LP ES LS 0.00 EP LP ES LS Ucn2 mrna expression showed the highest expression in the late secretory yphase Ucn3 mrna expression did not change during endometrial cycle Submitted 2010
32 Urocortin2 and inflammation Urocortin 2 Intestinal tract Placenta Proinflammatory action Moss et al. Gut 2007 Antiinflammatory action Proinflammatory action Submitted 2010
33 Urocortin 3 and inflammation Urocortin 3 Intestinal tract Saruta et al., Peptides 2005 Placenta Submitted 2010 Antiinflammatory Action
34 Effect of Ucn2 in cultured endometrial stromal cells on cytokines secretion 200 * ** TNF- pg/ml Control U2-8 U2-7 U2-6 U2-77 +ast2b U2-66 +ast2b Ucn2 treatment increased TNF- release in a dose dependent manner Astressin 2b reversed Ucn2 effects Submitted 2010
35 Effect of Ucn3 in cultured endometrial stromal cells on cytokines secretion L-4 pg/ml 2 1 ** *** * I 0 U U U Ast2b Ucn3 treatment increased IL-4 release Such effect was reversed by astressin 2b Submitted 2010
36 Neuroendocrine elements in endometriosis CRH Urocortin CRH-R1R1 Decidualization Infertility
37 Neuroendocrine elements in endometriosis Urocortin 2 Urocortin 3 CRH-R2 R2 Inflammation Pain
38 Acknowledgements Laboratory group Clinicians
39 Key points of established knowledge 1. Endometrial dysfunction is a cause of infertility in endometriosis 2. Increased abortion rate in endometriosis is related to endometrial dysfunction 3. Estrogen/Progesterone act on endometrial functions through locally produced growth factors/peptides and the alteration of this paracrine actions occurs in endometriosis 4. Endometrial cells implant in peritoneum is driven by immunomodulatory factors, facilitating neoangiogenesis and decreasing apoptotic process 5. Ectopic endometrium expresses some of the growth factors/peptides functionally produced by eutopic endometrium, with an overfunction of proinflammatory pathways
40 Unresolved areas A. Increased estrogen activity or progesterone resistance remain to be demonstrated in a large scale study by gene mutations / polimorphism evaluations B. Has ectopic endometrium identical characteristics of the eutopic tissue? C. The use of endometrial cells or factors for the noninvasive diagnosis of endometriosis is under investigation D. The endometrial action of estroprogestins in the treatment of endometriosis is not yet defined
41 Further research questions (1) Epidemiology: Classification: Pathogenesis: it is not clear how is really endometriosis diffuse in the Developing Countries is deep infiltrating endometriosis a separate disease or just a developmental stage? How critical are the serum estrogen/progesterone level changes in creating the endometrial substrate for endometriosis? How relevant are the epigenetic influences on estrogen/progesterone receptors? Is infertility related to specific endometrial pathways dysfunction? Diagnosis: Endometrial cytology may have a future for the diagnosis of endometriosis? Urinary or serum markers have sufficient specificity? Does uterine doppler analysis reveal some specific changes?
42 Further research questions (2) Treatment: Do surgical procedures contribute to spread endometriotic cells? Does it exist an optionally timing for the surgical treatment of endometriosis? Is induction of ovulation a treatment at risk for women with endometriosis? How to prevent the recurrency? Women s Health: How is the gestational outcome? How is he climateric period? How is the postmenopausal period in Women with endometriosis?
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