子宮內膜異位症是一個常見的婦科疾病，其病理特徵是子宮內膜組織出現在子宮腔以外的地方。高達30-50% 的子宮內膜異位症患者有不孕的困擾。然而，迄今為止，子宮內膜異位症所造成的不孕症的致病機轉仍未完全瞭解。初級纖毛是一種非運動性的胞器，由微管的軸絲、纖毛延伸膜和複雜的纖毛內運輸系統所組成。先前的研究發現初級纖毛與胚胎移植和植入等女性生殖有關。因此，我們認為初級纖毛可能通過調節子宮內膜對於胚胎的接受性進而造成子宮內膜異位症的女性有不孕的情形發生。首先，我們觀察正常的子宮內膜與有子宮內膜異位症的子宮內膜，發現在患者的子宮內膜組織中，有初級纖毛的子宮內膜基質細胞的百分比和初級纖毛長度比正常組織來的少。除此之外，我們分析了女性腹膜液對於子宮內膜基質細胞的纖毛新生所造成的影響。實驗結果發現子宮內膜異位症患者的腹腔液有抑制纖毛新生的趨勢。轉化生長因子-β1 (TGF-β1) 是腹膜液中高濃度的促炎細胞因子之一，已被證實會調控女性生殖，包括卵泡發育和激活、卵母細胞質量、排卵和子宮內膜蛻膜化，所以我們假設 TGF-β1 可能是抑制纖毛生長的關鍵因素。在研究中，我們觀察到在用 TGF-β1 處理的正常子宮內膜基質細胞中有初級纖毛的細胞百分比以及初級纖毛的長度顯著降低，而且我們發現由 TGF-β1所抑制的纖毛新生是通過增加纖毛新生相關基因-組蛋白去乙酰化酶 2 (HDAC2)。此外，子宮內膜蛻膜化對於成功的生育是不可或缺的。因此我們將子宮內膜基質細胞進行蛻膜化並給予TGF-β1，觀察TGF-β1是否會影響蛻膜化並分析HDAC2的表現量。結果發現用 TGF-β1 處理的細胞其蛻膜化的程度減少，並且增加了蛻膜細胞中 HDAC2 的表達。我們也建立子宮內膜異位症小鼠模型，計數子宮內膜組織中有初級纖毛的細胞數量以及測量纖毛的長度，結果顯示子宮內膜異位症小鼠的有纖毛的細胞百分比和纖毛長度顯著降低。綜合以上所述，我們發現子宮內膜異位症患者的腹膜液中升高的TGF-β1可能通過增強 HDAC2 的表達來減弱子宮內膜基質細胞的纖毛生成來降低子宮內膜對於胚胎的接受性。

Endometriosis is a gynecological disease which is characterized as the presence of endometriotic lesions outside of the uterine cavity. As high as 30-50% of patients with endometriosis are suffered from the infertile problems; however, the mechanism involved in endometriosis-associated infertility remains unclarified so far. Primary cilium is a non-motile organelle composed by a microtubule-based axoneme, ciliary extension membrane and complicated intraflagellar transporting system. Previous studies reveal that primary cilia are associated with female reproduction such as embryo transfer and implantation; therefore, we suggest that it may play a role in endometriosis-related infertility through modulating endometrial receptivity. First, we observed the percentage of ciliated cells and length of cilia were reduced in endometriotic lesion as compared to normal endometrium. Moreover, we analyzed the effect of women’s peritoneal fluid on ciliogenesis in endometrial stromal cells. The results indicated that treatment with peritoneal fluids from women with endometriosis inhibited ciliogenesis. Transforming growth factor-β1 (TGF-β1) is one of the highly-enriched pro-inflammatory cytokines in peritoneal fluids and has been reported to participate in several reproductive events including follicular development and activation, oocyte quality, ovulation, and decidualization. Therefore, we hypothesized that TGF-β1 may be the critical factor to inhibit ciliogenesis. Indeed, we observed that the percentage of ciliated cells as well as the length of primary cilia was significantly decreased in normal endometrial stromal cells treated with TGF-β1. Furthermore, we found the repressed ciliogenesis caused by TGF-β1 is via the enrichment of the ciliogenesis-related gene, histone deacetylase 2 (HDAC2), which has been reported to inhibit primary cilia formation in pancreatic ductal adenocarcinoma cells and is a target gene of Sma-and Mad-related protein 2 (SMAD2). Endometrium decidualization is indispensable to a successful fertility; therefore, we compared HDAC2 expression in decidual cells treated with or without TGF-β1. The results revealed that treatment with TGF-β1 increased HDAC2 expression in decidual cells. The mouse model of endometriosis was created and the frequency and length of cilia of uterus were measured. Results showed percent of ciliated cells and length of cilia were significantly reduced in uteri of endometriosis-like mice. Taken together, we showed that elevated TGF-β1 in peritoneal fluids in patients with endometriosis may reduce uterine receptivity through enhancing the expression of HDAC2 to attenuate ciliogenesis in endometrial stromal cells.

1. Czyzyk, A., et al., Update on endometriosis pathogenesis. Minerva Ginecol, 2017.
69(5): p. 447-461.
2. Giudice, L.C. and L.C. Kao, Endometriosis. Lancet, 2004. 364(9447): p. 1789-99.
3. Shafrir, A.L., et al., Risk for and consequences of endometriosis: A critical epidemiologic review. Best Pract Res Clin Obstet Gynaecol, 2018. 51: p. 1-15.
4. Agarwal, S.K., et al., Clinical diagnosis of endometriosis: a call to action. Am J
Obstet Gynecol, 2019. 220(4): p. 354 e1-354 e12.
5. Rolla, E., Endometriosis: advances and controversies in classification, pathogenesis, diagnosis, and treatment. F1000Res, 2019. 8.
6. Practice Committee of the American Society for Reproductive, M., Endometriosis
and infertility. Fertil Steril, 2004. 82 Suppl 1: p. S40-5.
7. Bulletti, C., et al., Endometriosis and infertility. J Assist Reprod Genet, 2010. 27(8):
p. 441-7.
8. Schenken, R.S., et al., Etiology of infertility in monkeys with endometriosis:
luteinized unruptured follicles, luteal phase defects, pelvic adhesions, and
spontaneous abortions. Fertil Steril, 1984. 41(1): p. 122-30.
9. D'Hooghe, T.M., L. Xiao, and J.A. Hill, Cytokine profiles in autologous peritoneal
fluid and peripheral blood of women with deep and superficial endometriosis. Arch
Gynecol Obstet, 2001. 265(1): p. 40-4.
10. Gazvani, R. and A. Templeton, Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction, 2002. 123(2): p. 217-26.
11. Iwabe, T., et al., Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil Steril, 1998. 69(5): p. 924-930.
12. Kalu, E., et al., Cytokine profiles in serum, and peritoneal fluid from infertile women with and without endometriosis. J Obstet Gynaecol Re, 2007. 33(4): p. 490-495.
13. Koninckx, P.R., S.H. Kennedy, and D.H. Barlow, Endometriotic disease: the role of
peritoneal fluid. Hum Reprod Update, 1998. 4(5): p. 741-751.
14. Maas, J.W.M., et al., Tumor necrosis factor-α but not interleukin-1β or interleukin-8 concentrations correlate with angiogenic activity of peritoneal fluid from patients with minimal to mild endometriosis. Fertil Steril, 2001. 75(1): p. 180-185.
15. Suginami, H. and K. Yano, An ovum capture inhibitor (OCI) in endometriosis
peritoneal fluid: an OCI-related membrane responsible for fimbrial failure of ovum
capture. Fertil Steril, 1988. 50(4): p. 648-53.
16. Lebovic, D.I., M.D. Mueller, and R.N. Taylor, Immunobiology of endometriosis.
Fertil Steril, 2001. 75(1): p. 1-10.
17. Kotlyar, A., H.S. Taylor, and T.M. D'Hooghe, Use of immunomodulators to treat
endometriosis. Best Pract Res Clin Obstet Gynaecol, 2019. 60: p. 56-65.
18. Christodoulakos, G., et al., Pathogenesis of endometriosis: the role of defective
'immunosurveillance'. Eur J Contracept Reprod Health Care, 2007. 12(3): p. 194-202.
19. Oosterlynck, D.J., et al., Women with Endometriosis Show a Defect in Natural-Killer Activity Resulting in a Decreased Cytotoxicity to Autologous Endometrium. Fertil Steril, 1991. 56(1): p. 45-51.
20. Oosterlynck, D.J., et al., The Natural-Killer Activity of Peritoneal-Fluid
Lymphocytes Is Decreased in Women with Endometriosis. Fertil Steril, 1992. 58(2):
p. 290-295.
21. Henriet, P., H.P. Gaide Chevronnay, and E. Marbaix, The endocrine and paracrine control of menstruation. Mol Cell Endocrinol, 2012. 358(2): p. 197-207.
22. Petracco, R.G., et al., Global gene expression profiling of proliferative phase
endometrium reveals distinct functional subdivisions. Reprod Sci, 2012. 19(10): p.
1138-45.
23. Maslar, I.A. and D.H. Riddick, Prolactin production by human endometrium during the normal menstrual cycle. Am J Obstet Gynecol, 1979. 135(6): p. 751-4.
24. Daly, D.C., I.A. Maslar, and D.H. Riddick, Prolactin production during in vitro
decidualization of proliferative endometrium. Am J Obstet Gynecol, 1983. 145(6): p.
672-8.
25. Jabbour, H.N. and H.O. Critchley, Potential roles of decidual prolactin in early
pregnancy. Reproduction, 2001. 121(2): p. 197-205.
26. Bulun, S.E., et al., 17Beta-hydroxysteroid dehydrogenase-2 deficiency and
progesterone resistance in endometriosis. Semin Reprod Med, 2010. 28(1): p. 44-50.
27. Hagiwara, H., N. Ohwada, and K. Takata, Cell Biology of Normal and Abnormal
Ciliogenesis in the Ciliated Epithelium, in International Review of Cytology. 2004,
Academic Press. p. 101-141.
28. Avasthi, P. and W.F. Marshall, Stages of ciliogenesis and regulation of ciliary length. Differentiation; research in biological diversity, 2012. 83(2): p. S30-S42.
29. Satir, P. and S.T. Christensen, Overview of Structure and Function of Mammalian
Cilia. Annual Review of Physiology, 2007. 69(1): p. 377-400.
30. Anvarian, Z., et al., Cellular signalling by primary cilia in development, organ
function and disease. Nature reviews. Nephrology, 2019. 15(4): p. 199-219.
31. Malicki, J.J. and C.A. Johnson, The Cilium: Cellular Antenna and Central Processing Unit. Trends Cell Biol, 2017. 27(2): p. 126-140.
32. Ishikawa, H., et al., Odf2-deficient mother centrioles lack distal/subdistal
appendages and the ability to generate primary cilia. Nat Cell Bio, 2005. 7(5): p. 517-524.
33. Graser, S., et al., Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol, 2007. 179(2): p. 321-30.
34. Singla, V., et al., Ofd1, a human disease gene, regulates the length and distal
structure of centrioles. Dev Cell, 2010. 18(3): p. 410-24.
35. Deane, J.A., et al., Localization of intraflagellar transport protein IFT52 identifies
basal body transitional fibers as the docking site for IFT particles. Curr Biol, 2001.
11(20): p. 1586-90.
36. Garcia-Gonzalo, F.R. and J.F. Reiter, Scoring a backstage pass: Mechanisms of
ciliogenesis and ciliary access. J Cell Biol, 2012. 197(6): p. 697-709.
37. Pedersen, L.B., et al., Assembly of primary cilia. Dev Dyn, 2008. 237(8): p. 1993-
2006.
38. Iomini, C., et al., Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases. J Cell Biol, 2001. 153(1): p. 13-24.
39. Marshall, W.F. and J.L. Rosenbaum, Intraflagellar transport balances continuous
turnover of outer doublet microtubules: implications for flagellar length control. J
Cell Biol, 2001. 155(3): p. 405-14.
40. Kobayashi, T., et al., Centriolar kinesin Kif24 interacts with CP110 to remodel
microtubules and regulate ciliogenesis. Cell, 2011. 145(6): p. 914-25.
41. Tsang, W.Y., et al., CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Dev Cell, 2008. 15(2): p. 187-97.
42. Broekhuis, J.R., W.Y. Leong, and G. Jansen, Regulation of cilium length and
intraflagellar transport. Int Rev Cell Mol Biol, 2013. 303: p. 101-38.
43. Kim, S., et al., Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry.Nat Cell Biol, 2011. 13(4): p. 351-60.
44. Li, A., et al., Ciliary transition zone activation of phosphorylated Tctex-1 controls
ciliary resorption, S-phase entry and fate of neural progenitors. Nat Cell Biol, 2011.
13(4): p. 402-11.
45. Pugacheva, E.N., et al., HEF1-Dependent Aurora A Activation Induces Disassembly of the Primary Cilium. Cell, 2007. 129(7): p. 1351-1363.
46. Pugacheva, E.N., et al., HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell, 2007. 129(7): p. 1351-63.
47. Kinzel, D., et al., Pitchfork regulates primary cilia disassembly and left-right
asymmetry. Dev Cell, 2010. 19(1): p. 66-77.
48. Hubbert, C., et al., HDAC6 is a microtubule-associated deacetylase. Nature, 2002. 417(6887): p. 455-8.
49. Lam, H.C., et al., Histone deacetylase 6-mediated selective autophagy regulates
COPD-associated cilia dysfunction. J Clin Invest, 2013. 123(12): p. 5212-5230.
50. Kasahara, K., et al., Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nat Commun, 2014. 5(1): p. 5081.
51. Nachury, M.V., et al., A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell, 2007. 129(6): p. 1201-13.
52. Boehlke, C., et al., Primary cilia regulate mTORC1 activity and cell size through
Lkb1. Nat Cell Biol, 2010. 12(11): p. 1115-22.
53. Goetz, S.C. and K.V. Anderson, The primary cilium: a signalling centre during
vertebrate development. Nat Rev Genet, 2010. 11(5): p. 331-44.
54. Satir, P., L.B. Pedersen, and S.T. Christensen, The primary cilium at a glance. J Cell Sci, 2010. 123(Pt 4): p. 499-503.
55. Ezratty, E.J., et al., A role for the primary cilium in Notch signaling and epidermal
differentiation during skin development. Cell, 2011. 145(7): p. 1129-41.
56. Lienkamp, S., A. Ganner, and G. Walz, Inversin, Wnt signaling and primary cilia.
Differentiation, 2012. 83(2): p. S49-55.
57. Christensen, S.T., et al., Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling. J Pathol, 2012. 226(2): p. 172-84.
58. Chegini, N., L.I. Gold, and R.S. Williams, Localization of transforming growth factor beta isoforms TGF-beta 1, TGF-beta 2, and TGF-beta 3 in surgically induced
endometriosis in the rat. Obstet Gynecol, 1994. 83(3): p. 455-61.
59. Pizzo, A., et al., Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol Obstet Invest, 2002. 54(2): p. 82-7.
60. Young, V.J., et al., Transforming growth factor-beta induced Warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis. J Clin Endocrinol Metab, 2014. 99(9): p. 3450-9.
61. Oosterlynck, D.J., et al., Transforming Growth-Factor-Beta Activity Is Increased in Peritoneal-Fluid from Women with Endometriosis. Obstet Gynecol, 1994. 83(2): p. 287-292.
62. Clement, C.A., et al., TGF-beta signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep, 2013. 3(6): p. 1806-14.
63. Lin, S.C., et al., Targeting Anthrax Toxin Receptor 2 Ameliorates Endometriosis
Progression. Theranostics, 2019. 9(3): p. 620-632.
64. Knowles, M.R., et al., Primary ciliary dyskinesia. Recent advances in diagnostics,
genetics, and characterization of clinical disease. Am J Respir Crit Care Med, 2013.
188(8): p. 913-22.
65. Barr, M.M. and P.W. Sternberg, A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature, 1999. 401(6751): p. 386-9.
66. Wheway, G., D.A. Parry, and C.A. Johnson, The role of primary cilia in the
development and disease of the retina. Organogenesis, 2014. 10(1): p. 69-85.
67. Brown, J.M. and G.B. Witman, Cilia and Diseases. Bioscience, 2014. 64(12): p.
1126-1137.
68. Sikora, J., et al., The involvement of multifunctional TGF-beta and related cytokines in pathogenesis of endometriosis. Immunol Lett, 2018. 201: p. 31-37.
69. Tarokh, M., et al., Serum and Peritoneal Fluid Cytokine Profiles in Infertile Women with Endometriosis. Iran J Immunol, 2019. 16(2): p. 151-162.
70. Wrana, J.L., et al., Mechanism of activation of the TGF-beta receptor. Nature, 1994. 370(6488): p. 341-7.
71. Kobayashi, T., et al., HDAC2 promotes loss of primary cilia in pancreatic ductal
adenocarcinoma. EMBO Rep, 2017. 18(2): p. 334-343.
72. Li, L., et al., MAP4 as a New Candidate in Cardiovascular Disease. Front Physiol,
2020. 11: p. 1044.
73. Ghossoub, R., et al., Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary cilium and control ciliary length. J Cell Sci, 2013. 126(Pt 12): p. 2583-94.
74. Zahnleiter, D., et al., MAP4-dependent regulation of microtubule formation affects centrosome, cilia, and Golgi architecture as a central mechanism in growth
regulation. Hum Mutat, 2015. 36(1): p. 87-97.
75. Follit, J.A., et al., The intraflagellar transport protein IFT20 is associated with the
Golgi complex and is required for cilia assembly. Mol Biol Cell, 2006. 17(9): p. 3781-92.
76. Follit, J.A., et al., The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi
complex. PLoS Genet, 2008. 4(12): p. e1000315.
77. Zhang, Z., et al., Intraflagellar transport protein IFT20 is essential for male fertility and spermiogenesis in mice. Mol Biol Cell, 2016.
78. Malutan, A.M., et al., Pro-inflammatory cytokines for evaluation of inflammatory
status in endometriosis. Cent Eur J Immunol, 2015. 40(1): p. 96-102.
79. Ni, N. and Q. Li, TGFbeta superfamily signaling and uterine decidualization. Reprod Biol Endocrinol, 2017. 15(1): p. 84.
80. Lin, S.C., et al., Suppression of COUP-TFII by proinflammatory cytokines
contributes to the pathogenesis of endometriosis. J Clin Endocrinol Metab, 2014.
99(3): p. E427-37.
81. Petit, F.G., et al., Deletion of the orphan nuclear receptor COUP-TFII in uterus leads to placental deficiency. Proc Natl Acad Sci U S A, 2007. 104(15): p. 6293-8.
82. Zhao, K.Q., et al., Maternal Smad3 deficiency compromises decidualization in mice. J Cell Biochem, 2012. 113(10): p. 3266-75.
83. Anvarian, Z., et al., Cellular signalling by primary cilia in development, organ
function and disease. Nat Rev Nephrol, 2019. 15(4): p. 199-219.
84. Colon-Diaz, M., et al., HDAC1 and HDAC2 are differentially expressed in
endometriosis. Reprod Sci, 2012. 19(5): p. 483-92.
85. Hrzenjak, A., et al., Valproate inhibition of histone deacetylase 2 affects
differentiation and decreases proliferation of endometrial stromal sarcoma cells.
Mol Cancer Ther, 2006. 5(9): p. 2203-10.
86. Fakhry, H., et al., Immunohistochemical detection of histone deacetylases in
endometrial carcinoma: involvement of histone deacetylase 2 in the proliferation of endometrial carcinoma cells. Hum Pathol, 2010. 41(6): p. 848-58.