The early life of endometriosis: lessons learned from the baboon

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3/26/29 The early life of endometriosis: lessons learned from the baboon Julie Hastings Human Reproductive Sciences Unit, MRC ESHRE, Glasgow, 29 Acknowledgments UIC Asgi Fazleabas Adrienne Olson

1 3/26/29 The early life of endometriosis: lessons learned from the baboon Julie Hastings Human Reproductive Sciences Unit, MRC ESHRE, Glasgow, 29 Acknowledgments UIC Asgi Fazleabas Adrienne Olson Andrea Braundmeier Kevin Jackson Mark Olson Patty Mavrogianis UCSF Linda Giudice Amy Hamilton Said Talbi Northwestern University Julie Kim Yale Hugh Taylor Jennifer Sarno University of Cincinnati SK Dey Susanne Tranguch Shuk-Mei Ho Xiang Zhang Shanghai Jiao Tong Univ Sun-Wei Guo Greenville Hospital Bruce Lessey Angela Howling UNC Steve Young Supported by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54 HD 493 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research What is Endometriosis? The presence of hormone-responsive endometrial tissue at ectopic sites beyond the uterine cavity Associated with chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility Affects in women of reproductive age Average time to diagnosis is 8- years Estimated economic burden of ~$22 billion (in 22) 2. Sinaii et al., 28. Fertil Steril;89: Simoens et al., 27. Hum Reprod Update; 3:395-44

s theory of retrograde menstruation. Sampson, 927.

2 3/26/29 How does Endometriosis Develop? Halban s theory of hematogenic or lymphogenic spread of viable endometrial cells Meyer s theory of coelomic metaplasia of peritoneal mesothelial lining Sampson s theory of retrograde menstruation. Sampson, 927. Am J Obstet Gynecol;4: Limitations in Understanding the Pathophysiology of Endometriosis 9% of women experience retrograde menstruation Only % of women develop endometriosis 3-5% of women with endometriosis i are infertile What is the pathophysiology of endometriosis-associated infertility? Is Endometriosis Associated with Infertility? Barnhart et al., 22. Fertil Steril; 77:

3 3/26/29 Does Lesion Ablation Improve Fertility? Marcoux et al., 997. N Engl J Med; 337: Parazzini, 999. Hum Reprod; 4: Limitations in Understanding the Pathophysiology of Endometriosis Does the presence of endometriotic lesions affect reproductive/endometrial function? OR Does an inherently defective endometrium affect reproductive function and the growth of endometriotic lesions? Limitations in Understanding the Pathophysiology of Endometriosis Disease has been present for an extended period at the time of diagnosis Availability of endometrial tissue during the receptive phase is limited Clinical studies with appropriate controls are not feasible 3

3/26/29 Experimental Models of Endometriosis in the Non-human Primate Spontaneous endometriosis reported in the macaque and baboon families of monkeys -4 Surgical diversion of the cervix into the

and experimental endometriosis 9. McClure et al., 97. J Med Assoc Ga;6:-3 2. Fanton et al., 983. Lab Anim Sci;33:597-599 3. Digiacomo et al., 977. J Am Vet Med Assoc;7:859-86 4. Merrill, 968.

D Hooghe et al., 994. Fertil Steril;62:635-638 8. D Hooghe et al., 995.

4 3/26/29 Experimental Models of Endometriosis in the Non-human Primate Spontaneous endometriosis reported in the macaque and baboon families of monkeys -4 Surgical diversion of the cervix into the abdomen resulted in endometriosis 5-7 Intra-peritoneal seeding with autologous menstrual endometrium in baboons results in endometriosis 8 Fecundity was reduced in baboons with advanced spontaneous and experimental endometriosis 9. McClure et al., 97. J Med Assoc Ga;6:-3 2. Fanton et al., 983. Lab Anim Sci;33: Digiacomo et al., 977. J Am Vet Med Assoc;7: Merrill, 968. Am J Obstet Gynecol;: D Hooghe et al., 26. Fertil Steril; 66: Te Linde and Scot, 95. Am J Obstet Gynecol;6:47-73, 6. Allen et al., 954. Am J Obstet Gynecol;68: D Hooghe et al., 994. Fertil Steril;62: D Hooghe et al., 995. Am J Obstet Gynecol;73:25-34 Experimental Induction of Endometriosis in the Baboon Laparoscopy And Inoculation With Autologous Menstrual Laparoscopic Evaluation of Disease Harvest of Eutopic Endometrium by Laparotomy During Window of Receptivity Tissue 3 m 6 m 9 m 2m 5 m Experimental Endometriosis Recapitulates Spontaneous Disease Induced Disease in the Baboon Spontaneous Human Disease 4

5 3/26/29 Pathological Diagnosis of Endometriosis Chocolate Lesion on the uterus Red/Blue Lesion on the uterus Blue Lesion on the uterus Blue Lesion in peritoneal fat Chocolate Lesion on the bladder Red Lesion on the body wall Hastings and Fazleabas 26. Reprod Biol Endocrinol;4 (Suppl):S7 Characterization of Lesions Visualized by Laparoscope in Baboons Experimentally Induced with Endometriosis 8 Number of 7 Lesions 6 5 Red Blue Chocolate White Mixed month (n=6) 3 mon (n=2) 6 mon (n=) 9 mon (n=6) 2 mon (n=9) 5 mon (n=5) Necropsy (n=6) Is endometriosis-associated infertility mediated, in part, by a defective endometrium? 5

3/26/29 Affymetrix Microarray Analysis in Baboons with Endometriosis Laparoscopy And Inoculation With Autologous Menstrual Laparoscopic Evaluation of Disease Harvest of Eutopic Endometrium by

6 3/26/29 Affymetrix Microarray Analysis in Baboons with Endometriosis Laparoscopy And Inoculation With Autologous Menstrual Laparoscopic Evaluation of Disease Harvest of Eutopic Endometrium by Laparotomy During Window of Receptivity Tissue mth 3 m 6 m 9 m 2m 5 m Hybridisation Efficiency of Baboon cdna to HG U33 Array Probe Set Count Number Present Number Absent Number Marginal Filtered Hierarchical Clustering* mont 3 mont 3 mont 3 mont nth 2 mont mon nth mon 2 mont 2 mont ous ous Spontaneo Spontaneo 6 mont 5 mont 5 mont 5 mont rol Contr 6 mont 6 mont rol rol Cont Cont *Probe sets (687) that are differentially expressed between controls and, 3, 6, 2, and 5 mon of experimental disease or spontaneous disease. (Biological and hybridization outliers are excluded.) 6

7 3/26/29 Time Course of Gene Dysregulation Month 3 Mon 6 Mon 2 Mon 5 Mon Spontaneous Up-regulated Down-regulated Time Course of Gene Dysregulation No correlation with pain 25 Lesion Number 2 5 Total New No correlation with total lesion or new lesion load No correlation with lesion type No correlation with lesion position 5 Lesion Number Red Chocolate Blue White Mixed Uterus Lesion Cul de Sac Number 5 Bladder 4 Peritoneal Wall Rectovaginal Pouch 3 Omentum Mon post inoculation Comparison of Gene Dysregulation Up-regulated Down-regulated month 3 mon 453 (9%) 69 (3%) 6 mon 4 (.2%) 6 (.8%) 2 mon 45 (7%) 53 (26%) 5 mon 5 (.2%) 24 (%) Spontaneous 289 (2%) 45 (7%) 3 mon 6 mon 6 (.8%) (.3%) 2 mon 27 (29%) 375 (45%) 5 mon (.%) 3 (2%) Spontaneous 9 (5%) 67 (8%) 6 mon 2 mon 2 (4%) 8 (3%) 5 mon 2 (4%) (%) Spontaneous 5 (29%) (2%) 2 mon 5 mon 6 (.7%) 2 (2%) Spontaneous 2 (6%) 64 (9%) 7

8 3/26/29 Common Up-regulated Genes at, 3, & 2 Mon of Disease Month 3 Mon Mon Common Down-regulated Genes at, 3, & 2 Mon of Disease Month 3 Mon Mon Gene Ontology Classification Selected Pathways Regulated by Endometriosis EGF-R signaling pathway TGFβ-R signaling pathway WNT signaling pathway Olfactory receptor activity Rho protein signal transduction Ras GTPase activity/signal transduction Angiogenesis/vasculogenesis Chemokine/cytokine activity Immune response/inflammatory response Chromatin assembly/disassembly Methyltransferase/acetyltransferase activity Chromatin binding Microtubule cytoskeleton organization and biogenesis Cell adhesion/cell matrix/extracellular matrix Regulation of cell growth/proliferation Nervous system development GTPase signal transduction 8

9 3/26/29 Commonly Dysregulated Genes Induced vs. Human Endometriosis Month 3 Mon 2 Mon ABCB6 KCNJ4 HIPK3 CDKN2B ALDH6A CARD AGRN CDKN2B MEG3 JAG JAG CENTB CDKN2B BBP JAG CDKN2B CARD MGAT3 MGAT3SUCGL2 KIAA256 CHRNE MGAT3 FZD6 SUCGL2 MEG3 MGAT3 CD79A MUC3B MSC CDKN2B RIN3 MUC3B MAP7 PICALM COLA MUC3B KCNJ4 RIN3 HIPK3 JAG MGAT3 MUC3B MSC RIN3 CENTB PDE4A RAB33B MGAT3 SUCLG2 MUC3B CHRNE RGR SERPINB9 MUC3B RGR COLA RIN3 SUCGL2 RIN3 RIN3 DGCR8 SFTPC TMEFF TCF3 TMEPAI EDIL3 SHANK UTX ZNF25 GSK3A TCF5 HRK TCF3 IGFALS TPARL IGLJ3 ZNF25 BBP JAG SUCGL2 Burney et al, 27. Endocrinol;48: Coordinated Endometrial Changes Associated with Endometriosis FOS FOS FOS CYR6 2 CYR6 CYR6 EMMPRIN 3 EMMPRIN EMMPRIN ER 4 ER ER ` PR 4 PR PR Calcitonin 5 Calcitonin Calcitonin FKBP52 4 FKBP52 FKBP52 HOXA 6 HOXA HOXA Inoculation Progression of Disease (mon). Hastings et al, 26. BOR;75: Jackson et al, 27. Reprod Sci; 4: Gashaw et al, 26. BOR;74: Jackson et al, 27. Reprod Sci; 4 (Suppl):A62 3. Braundmeier et al. SSR 26; Abstract #27 6. Kim et al., 27. Mol Hum Reprod;3: FOS is Transiently Increased in Baboons with Endometriosis 6mth disease 5mth disease Relative mrna Expression * 5μm 5 2μm 5 3 mon Kruskal Wallis ANOVA, p-value=.38 * Dunn's Correction for multiple testing, p-value<.5 * 6 mon 9 mon 2 mon 5 mon Hastings et al, 26. BOR;75:

10 3/26/29 EMMPRIN is Transiently Increased in Baboons with Endometriosis 3 EMMPRIN mrna Fold Change 2 Kruskal Wallis ANOVA, p<.5 Mth 3 M 9 M Braundmeier, unpublished HOXA is Decreased in Baboons with Endometriosis 5 month Relative 4 HOXA Expression 3 2 *Kruskal Wallis ANOVA, p<.5 * 3 mon 6 mon 2 mon 5 mon * Kim et al, 27 Mol Hum Reprod;3: SHRs are Altered in Baboons with Endometriosis Eutopic 3 mon Endometriotic Eutopic 5 mon Endometriotic Eutopic ESR PGR Jackson et al, 27. J Reprod Sci;4(2):37-5

11 3/26/29 FKBP52 is Decreased in Baboons with Endometriosis 6mth disease 5mth disease 6 mth disease 5 mth disease FKBP52 β-actin Jackson et al., Reprod Sci 4:37, 27 FKBP52 is Critical for Uterine Receptivity HOXA LTF +/+ -/- Tranguch et al., 25. PNAS; 2: FKBP52 is Decreased in Baboons with Endometriosis 6mth disease 6 mth disease 5 mth disease Endometriosis Spontaneous Induced FKBP52 β-actin Pgr-B Pgr-A E, P + camp Jackson et al., Reprod Sci 4:37, 27

12 3/26/29 Coordinated Endometrial Changes Associated with Endometriosis FOS FOS FOS CYR6 2 CYR6 CYR6 EMMPRIN 3 EMMPRIN EMMPRIN ER 4 ER ER ` PR 4 PR PR Calcitonin 5 Calcitonin Calcitonin FKBP52 4 FKBP52 FKBP52 HOXA 6 HOXA HOXA Inoculation Progression of Disease (mon). Hastings et al, 26. BOR;75: Jackson et al, 27. Reprod Sci; 4: Gashaw et al, 26. BOR;74: Jackson et al, 27. Reprod Sci; 4 (Suppl):A62 3. Braundmeier et al. SSR 26; Abstract #27 6. Kim et al., 27. Mol Hum Reprod;3: Summary Experimental endometriosis in the baboon closely recapitulates the human disease Induction of disease has an immediate effect on the transcriptome of the eutopic endometrium Endometriosis temporally regulates gene expression The presence of peritoneal endometriotic lesions impact the transcriptome of the eutopic endometrium How does endometriosis mediate this massive gene dysregulation? 2

13 3/26/29 FOS is Increased in Baboons with Endometriosis 3 mrna 6mth disease 5mth disease Level * μm 5 2μm 5 * * Kruskal-Wallis ANOVA, p-value=.7 *Dunn s Correction for Multiple testing, p-value>.5 * 3m 6m 5m control lesions control disease control disease Eutopic Endometrium Peritoneum Ovary Hastings et al, 26. BOR;75:76-82 The Role of FOS in the Endometrium Murine uterine mrna specifically induced by E2,2 Mediates murine estrogenic proliferative response,2,3 FOS protein and mrna levels are maximal in human proliferative endometrium 4 FOS protein and mrna levels are completely suppressed by MPA in vivo 5 FOS inhibits the transcriptional activity of PR 6 GnRH decreased c-fos mrna in endometrial cells in vitro 7. Nephew et al., 993. J Steroid Biochem Mol Biol;46: Cicatiello et al., 993. Receptor;2: Mendoza-Rodriguez et al., Mol Reprod Dev;64: Reis et al, 999. Fertil Steril;7: Kirkland et al, 992. Endocrinol;3: Shemshedini et al, 99. EMBO J;: Luo et al, 24. Am J Physiol Endocrinol Met; 287: E99- FOS Regulates Methylation FOS induced transformation of fibroblast mediated by DNA methyltransferase (DNMT) DNMT inhibition of FOS partially reverses transformation -IPTG +IPTG dnmt 8s rrna Bakin and Curran, 999. Science;283:

methyltransferases (DNMT) establish/maintain i t i pattern of methylation Histone deacetylase

DNMT3a DNMT3b Yamagata et al, 29. doi:.93/humrep/dep5 Wu et al, 26.

14 3/26/29 What is Methylation? Epigenetic modification of DNA (genomic imprinting) Cytosine in CpG dinucleotides DNA methyltransferases (DNMT) establish/maintain i t i pattern of methylation Histone deacetylase (HDAC) mediates chromatin compaction Transcriptional silencing DNMT Enzymes in the Endometrium DNMT DNMT3a DNMT3b Yamagata et al, 29. doi:.93/humrep/dep5 Wu et al, 26. Fertil Steril;87:24-32 DNMT Is Increased in Baboons with Endometriosis 3 Month DNMT3a and DNMT3b were not expressed in baboon endometrium (normal or endometriotic) 6 DNMT 8S 5 2 Month 5 Mon mon 6 mon 9 mon 5 mon 4

15 3/26/29 5 CpG Islands in the HOXA Gene 3.7kb CpG Islands CpG Islands 5 -UTR ~.kb ~.2kb Exon Exon 2 CpG site Protein Coding Region Protein Non-coding Region CpG a b d c e f g i h j k l m n o p q r s t u v The HOXA Gene is Methylated in Baboons With Endometriosis Disease Disease Disease Disease Disease Disease Kim et al, 27 Mol Hum Reprod;3: F Fragment The HOXA Gene is Methylated in Women With Endometriosis Wu et al, 25. Am J Obstet Gynecol; 93:37-38 F2 Fragment F3 Fragment

16 3/26/29 ESR is Hypomethylated in Human Endometrioma Xue et al, 27. Biol Reprod: 77: Methylation Sensitive Restriction Fragmentation Digestion of Genomic DNA with Methylation Insensitive Restriction Enzyme (MseI) No Further Digestion Second Digest with Methylation Sensitive Restriction Enzyme (BstUI) Undigested Digested Methylated DNA Unmethylated DNA Amplification with pairs of Arbitrary Primers Fragments Hybridize Large Methylated Fragments Hybridize Small Unmethylated Fragments Do Not Hybridize MseI MseI MseI BstUI MseI MseI BstUI Differentially Methylated Fragments Sequenced and Aligned with Human and non/human Primate Sequence Databases Differentially Methylated Fragments in Baboon Endometriotic Endometrium Fragment Size Gene Homology Hypo-methylated Location of Fragment Gene Function or Description 43bp WNT2B 5 -UTR Growth & development 55bp TMEM97 5 -UTR Regulation of cell death 548bp OR4D 5 -UTR Olfactory receptor 36bp PLEKHG2 5 -UTR Rho signal transduction 77bp SHOX Intron/Exon Growth & development Fragment Size Gene Homology Hyper-methylated Location of Fragment Gene Function or Description 29bp LGALS9 5 -UTR Immune cell apoptosis 6

17 3/26/29 The Human WNT2B Gene is Hypomethylated in Endometriosis ATG CG% 8 CpG CG Islands ** * * Hypo-methylation Hotspots Exon Relative mrna Expression (x 5 ) Disease Endometriosis-Associated Infertility a working hypothesis Aberrant levels of enzymes involved in methylation Epigenetic modification of endometrial genome Altered SHR Profile Aberrant gene expression Cell transformation Non-responsive to the embryonic signal IMPLANTATION FAILURE 7

Endometrio ed endometriosi: the same tissue?

Endometrio ed endometriosi: the same tissue? Valentino Remorgida Clinica Ostetrica e Ginecologica IRCCS Azienda Ospedaliera Universitaria San Martino IST Istituto Nazionale per la Ricerca sul Cancro, Università