Pathogenesis of Pain in Endometriosis Serdar Bulun, MD George H. Gardner Professor of Clinical Gynecology Chief, Division of Reproductive Biology Research Department of Obstetrics and Gynecology Northwestern University, Chicago, IL

ENDOMETRIOSIS Common cause of chronic pelvic pain and infertility Polygenic inheritance and multifactorial etiology 3-6 million women of reproductive age are affected in the USA Responsible for 10%-15% of all hysterectomies performed in the USA

GLANDS STROMA

MEDICAL TREATMENT OPTIONS Oral contraceptives GnRH analogs Progestins Aromatase inhibitors Selective Progesterone Receptor Modulators

FAT AND SKIN ENDOMETRIOSIS adrenal OVARY aromatase inhibitor CHOLESTEROL ANDROSTENEDIONE StAR adrenal 3-Estradiol ANDROSTENEDIONE ESTRONE ANDROSTENEDIONE aromatase 1-Estradiol OVARY 2-Estradiol aromatase inhibitor aromatase GnRH-a OC FAT AND SKIN

E 1 2 COX-2 aromatase ESTROGEN COX-2 3 Endometrium from PRECURSOR Endometrium from Disease-free Women 1 COX-2 PRECURSOR PGE2 PGE2 SF1 PRECURSOR aromatase Endometrium from Women with Endometriosis ESTROGEN E 2 COX-2 PGE2 PRECURSOR PGE2 SF1 aromatase ESTROGEN PRECURSOR E COX-2 3 Endometriosis PRECURSOR PGE2 PGE2 Noble, et al, JCEM, 1996; Ota, et al, 2001

NUCLEAR RECEPTORS (NR) membrane receptor nonclassical NR P Src CYTOPLASM MAPK steroid receptor orphan nuclear receptor NR NR PROTEIN PRODUCTS OF TARGET GENES BIOLOGIC FUNCTION e.g., DIFFERENTIATION P classical NUCLEUS NR P NR P TF NRE Target gene DNA

(mRNA by real-time PCR) NUCLEAR RECEPTORS AND ENDOMETRIOSIS ENDOMETRIUM (TISSUE) OVARIAN ENDOMETRIOMA (TISSUE) 14.1 142.4 9.3 1.0 >12,000 8.9 10 0.8 9 8 7 0.6 (mRNA by real-time PCR) fold difference SF1 mRNA 6 5 0.4 4 3 0.2 2 1 ERβ Total PR PR-B E-IUM E-OSIS ERα Xu Q et al, BOR 2007

EPIGENETIC DEFECTS IN ENDOMETRIOSIS SF-1 HYPOMETHYLATION

Methylation of CpG islands at promoters and gene expression Unmethylated CpG site exon1 exon2 exon3 on off Promoter CpG island Methylated CpG site DNA methylation only affects the cysteines(C) that is followed by a guanosine(G) in DNA sequence. When the CpG dinucleotides clustered in small stretches of DNA, this is termed “ CpG island. If the CpG island located in the promoter region of a gene is methylated, the gene expression will be turned off, but if not methylated, the gene expression will be turned on.

SF1 GENE PROMOTER NORMAL SUBJECTS PATIENTS WITH ENDOMETRIOSIS

SF1 PROMOTER IS NOT METHYLATED IN ENDOMETRIOSIS Transcription SF1 ENDOMETRIOSIS coactivating on complex exon1 exon2 exon1 exon2 exon1 exon2 HDAC1 mSin3A SF1 ENDOMETRIUM off MeCP2 Methylation can directly interfere with the DNA binding of certain transcriptional factors, or some methyl-CpG binding proteins are able to change DNA conformation and thus affect the binding of various transcriptional regulators. exon1 exon2 exon1 exon2 exon1 exon2 unmethylated CpG island methylated CpG island Xu Q et al, JCEM 2007

ROLE OF SF-1 IN ESTROGEN PRODUCTION

PERITONEAL ENDOMETRIOSIS 20 40 60 80 100 120 140 160 E-IUM E-OSIS A * p<0.01 0.5 1 1.5 2.0 2.5 B E-IUM E-OSIS * C 0.05 0.1 0.15 0.2 0.25 0.3 0.35 D * p<0.05 p<0.001 p<0.01 * 0.7 0.6 0.5 StAR mRNA (amol/mg 18S) P450scc mRNA (amol/mg 18S) 3HSDBII (amol/mg 18S) P450c17 mRNA (amol/mg 18S) 0.4 0.3 0.2 0.1 E-IUM E-OSIS E-IUM E-OSIS E F G p<0.001 p<0.05 p<0.001 2.0 * 0.2 0.4 0.6 0.8 1.0 E-IUM E-OSIS * 4 1.5 3 P450arom mRNA (amol/mg 18S) * SF-1 mRNA (amol/mg 18S) LRH-1 mRNA (amol/mg 18S) 1 2 0.5 1 un- detectable E-IUM E-OSIS E-IUM E-OSIS Attar and Tokunaga et al, JCEM, 2009

OVARIAN ENDOMETRIOTIC STROMAL CELLS B P450scc (amol/mg 18S) 2 4 6 8 10 12 14 PGE2: - + - + E-OSIS *p= 0.048 * * 30 *p= 0.032 25 StAR (amol/mg 18S) 20 * * 15 10 5 PGE2: - + - + E-IUM E-OSIS E-IUM E-IUM C D 0.06 * * 0.25 0.05 * *p= 0.05 *p= 0.012 0.20 HSD3BI (amol/mg 18S) 0.04 HSD3BII (amol/mg 18S) 0.15 0.03 0.02 0.10 * 0.01 0.05 0.00 E-IUM E-IUM E-OSIS E-OSIS 0.00 E-IUM E-IUM E-OSIS E-OSIS PGE2: - + - + PGE2: - + - + E F 0.00 0.02 0.04 0.06 0.08 0.10 0.12 P450c17 (amol/mg 18S) * E-OSIS PGE2: - + - + P450arom (amol/mg 18S) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 * *p= 0.030 *p= 0.042 E-IUM E-IUM E-IUM E-IUM E-OSIS E-OSIS PGE2: - + - + Attar and Tokunaga et al, JCEM, 2009

ENDOMETRIOSIS nmol/L ++++ +++ ++ + pmol/L StAR P450scc HSD3B2 HO ENDOMETRIOSIS 6 1 2 3 4 5 9 7 8 10 11 12 13 14 15 16 17 18 19 21 20 22 23 25 24 27 26 cholesterol StAR HO C CH3 O P450scc O C CH3 MITOCHONDRION HSD3B2 nmol/L ++++ pregnenolone PROGESTERONE +++ ++ + P450c17 HO C CH3 O OH P450c17 O C CH3 OH HSD3B2 17-hydroxypregnenolone 17-hydroxyprogesterone P450c17 P450c17 HO O O O OH HSD3B2 HSD17B1 dehydroepiandrosterone androstenedione testosterone P450arom P450arom HO O HO OH HSD17B1 pmol/L estrone ESTRADIOL

estradiol ++ PGE2 COX-2 ++ ++ ++ aromatase aromatase inhibitor ENDOMETRIOSIS TISSUE ++ PGE2 AA COX-2 ++ SF1 IL-1b VEGF ++ CHOLESTEROL StAR scc estradiol 3b-HSD-2 adrenal ++ 17-OH-lase 17/20-lyase aromatase inhibitor aromatase androstenedione estrone Noble et al, JCEM, 1997 Zeitoun et al, Mol Endo, 1999 Tamura et al, JBC, 2002 Tamura et al, JCEM, 2002 Attar and Tokunaga, et al, JCEM, 2009 Takayama et al, Fertil Steril, 1998 Ailawadi et al, Fertil Steril, 2004 Amsterdam et al, Fertil Steril, 2005

FSH LH + OC or P FSH LH FSH LH Postmenopausal on AI Follicle Development Prememopausal on AI (Hypothetical) Aromatase FSH LH E2 AI Peripheral Prememopausal on AI + OC or P Aromatase Peripheral AI OC P GnRHa E2 FSH LH Hypothalamus Aromatase Pituitary FSH LH No Follicular Aromatase Ovary AI Endometriosis Aromatase Peripheral Tissues Peripheral Aromatase Takayama et al, Fertil Steril, 1998; Ailawadi et al, Fertil Steril, 2004; Soysal et al, Human Reproduction, 2004; Amsterdam et al, Fertil Steril, 2005; Attar and Bulun Fertil Steril, 2006

Pre- and Post-treatment Pain Scores (n=10) PREMENOPAUSAL WOMEN TREATED WITH LETROZOLE AND NORETHINDRONE ACETATE Pre- and Post-treatment Pain Scores (n=10) 5 10 5 10 Average Pain Score p=0.0028 Baseline post-treatment Ailawadi, et al, Fertil Steril, 2004

PREMENOPAUSAL WOMEN TREATED WITH LETROZOLE AND NORETHINDRONE ACETATE Before Treatment Ailawadi, et al, Fertil Steril, 2004

PREMENOPAUSAL WOMEN TREATED WITH LETROZOLE AND NORETHINDRONE ACETATE Before Treatment Ailawadi, et al, Fertil Steril, 2004

PREMENOPAUSAL WOMEN TREATED WITH LETROZOLE AND NORETHINDRONE ACETATE After Treatment for 6 Months Ailawadi, et al, Fertil Steril, 2004

SIDE EFFECTS Mild hot flushes (2/day – 1/week) Sleepiness Mood swings Decreased libido No bone loss

TREATMENT RECOMMENDATIONS An aromatase inhibitor is the treatment of choice for persistent postmenopausal endometriosis A combination of an aromatase inhibitor with an OC or GnRH analog may be used to treat endometriosis-related pain not responding to conventional treatments Await results of randomized studies for the routine use of aromatase inhibitors for premenopausal endometriosis, uterine leiomyomata and ovulation induction

EPIGENETIC DEFECTS IN ENDOMETRIOSIS ERb HYPOMETHYLATION

Bisulfite sequencing: ERβ promoter 44481bp 10957bp TSS +1 Exon 0N Exon 0K Exon 0N Exon 1 Classical CpG island 556bp 100bp Bisulfite sequence NORMAL SUBJECTS PATIENTS WITH ENDOMETRIOSIS We identified an approximately 550-bp classical CpG island (-197/+359) within the promoter and exon І region of the ER-β gene. methylation status of ER-β promoter region was characterized by bisulfite genomic sequencing. For each subject, 6 to 8 clones were sequenced. Open and filled circles represent unmethylated and methylated cytosines, respectively. The numbers on the top indicate the positions of cytosine residues of CpGs relative to the transcription start site (+1), and the numbers, 1 to 8 on the left, represent different primary cultured stromal cells in the two groups. It was heavily methylated in most of endometrial stromal cells (n=8) that expressed lower level of ER-β and largely unmethylated in endometriotic stromal cells (n=8) that expressed higher level of ER-β mRNA. P< 0.001

+ ERb gene HDAC1 Pr 0N Pr 0K Pr 0N Pr 0K CBP CpG island CpG island Methylated HDAC1 Pr 0K Pr 0N CBP + mSin3a MeCP2 Unmethylated CpG island ENDOMETRIUM ENDOMETRIOSIS

ROLE OF ERb IN PROSTAGLANDIN PRODUCTION

PROSTAGLANDIN AND ESTRADIOL BIOSYNTHETIC PATHWAYS ANDROSTENEDIONE Aromatase ESTRONE ? PGE2 HSD17B1 mPGES-1 ESTRADIOL PGH2 COX-2 ARACHIDONIC ACID PLA2 PHOSPHOLIPIDS

PROSTAGLANDIN AND ESTRADIOL BIOSYNTHETIC PATHWAYS ANDROSTENEDIONE Aromatase ESTRONE PGE2 HSD17B1 mPGES-1 ESTRADIOL ERb PGH2 COX-2 ARACHIDONIC ACID IL-1b VEGF PLA2 PHOSPHOLIPIDS

ROLE OF ERb IN PROGESTERONE RESISTANCE

_ _ ++++ _ _ ++++ E2 + P E2 E2 E1: 17b-HSD-2: Secretory Menses endometrium Menses Proliferative endometrium _ _ E2 E1: ++++ _ _ ++++ 17b-HSD-2: (Epithelial) Bulun SE, et al, MCE, 2006

E2 E1 HSD17B2 + EUTOPIC ENDOMETRIUM E1 ENDOMETRIOSIS E2 PROGESTERONE HO OH HSD17B2 E2 HO E1 + EUTOPIC ENDOMETRIUM PROGESTERONE HO OH HO O E2 HSD17B2 E1 progesterone resistance ENDOMETRIOSIS PROGESTERONE Zeitoun, et al, JCEM, 1998 Yang, et al, Mol Endo, 2001

T47D Cycle Day: 3 6 10 12 14 16 18 24 26 PR-B PR-A PR-B PR-A EUTOPIC ENDOMETRIUM PR-A PR-B ENDOMETRIOSIS PR-A Attia, et al, JCEM, 2000

ENDOMETRIOSIS ERb: ERa ERa: gene + PR-B: PR-A: ERb gene PR gene HDAC1 Prom 0N CpG island MeCP2 HDAC1 mSin3a ENDOMETRIOSIS ERb gene ERb: ERb ERa gene Prom C Prom A ERa: PR gene PR-A PR-B ERa + ERb ERb PR-B: PR-A: Trukhacheva et al, JCEM, 2009

Bulun, SE, NEJM 2009 Cheng, Y, JCEM, 2008

BIG PICTURE

SF1 PGE2 COX-2 arom estradiol ERb PR ERa methylation HSD17B2 estrone CHOLESTEROL retinoic acid PR methylation ERb ERa Bulun et al, NEJM, 2009

Bulun, SE, NEJM 2009

FAILURE OF CELLS TO IMPLANT SURVIVAL AND IMPLANTATION OF NORMAL ENDOMETRIOSIS ROLE OF EPIGENETICS IN MECHANISM OF ENDOMETRIOSIS Painful menses Menses unmethylated promoter peritoneum peritoneum methylated promoter + SF1 ERb OTHERS FAILURE OF CELLS TO IMPLANT SURVIVAL AND IMPLANTATION OF ENDOMETRIOTIC CELLS CELLS CLEARED BY IMMUNE SYSTEM

BULUN LAB COLLABORATORS SUPPORT CURRENT: NICHD R37-38691 U54-40093 PREVIOUS: Jianfeng Zhou Li Meng Sijun Yang Juan Fang David Langoi Bilgin Gurates Siby Sebastian Mitsutoshi Tamura Khaled Zeitoun Kazuto Takayama George Attia Luis Noble Santanu Deb Gonca Imir Regina Martin Stephen Thung Veysel Fenkci Masashi Demura Sanober Amin Wen Cao Hiroki Utsunomiya Erkut Attar Bertan Yilmaz Newton Lee Amy Hakim Hideki Tokunaga Hiroshi Ishikawa Carrie Richardson Megan O’Hallaran BULUN LAB COLLABORATORS CURRENT: Chris Brooks John Coons Dong Chen Youhong Cheng Matthew Dyson Xia Luo Erica Marsh Diana Monsivais Irene Moy Antonia Navarro Mary Ellen Pavone Kerry Pearson Emily Su Ping Yin Hong Zhao Julie Kim Romana Nowak Debu Chakravarti Hironobu Sasano Takashi Suzuki Nobuo Yaegashi Kunihiro Okamura Evan Simpson Mitch Dowsett Seema Khan Michael Putman Asgi Fazleabas Alan Johns David Redwine Kirk Neely Berenice Mendonca Makio Shozu Erkut Attar NICHD R37-38691 U54-40093 SUPPORT