TGF-β1 Neutralization Improves Pregnancy Outcomes by Restoring Endometrial Receptivity in Mice with Adenomyosis

The objective of this research is to study the effects of TGF-β1 inhibition on endometrial receptivity and pregnancy outcomes in mice with adenomyosis. Experiments were done using a mouse model of adenomyosis which took place in a hospital-affiliated laboratory. The mouse model used for this research is ICR mouse. Adenomyosis was induced by oral gavage of tamoxifen (TAM) from postnatal days (PNDs) 1 to 4 in ICR mice. Bilateral intrauterine injection of anti-TGF-β1-neutralizing antibody or isotype IgG or PBS was performed at PND42. The mice were then either sacrificed or mated at PND64 followed by sacrificing at gestational day (GD) 4 or proceeding to delivery. Implantation numbers, rate of dams with live birth, live birth numbers, survival at 1 week old, and pup mortality rate after weaning were recorded. Collagen was demonstrated by Masson’s trichrome and Van Gieson’s stains. Uterine expression of a receptivity marker, leukemia inhibitory factor (LIF), was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC). Anti-TGF-β1 treatment increased the mean implantation numbers, fecundity rate, the rate of dams with live birth, pup survival rate at 1 week old, and pup mortality rate after weaning. Collagen expression in uteri with adenomyosis was attenuated by anti-TGF-β1 treatment. Increased LIF expression by anti-TGF-β1 treatment was detected by qRT-PCR, Western blot, and IHC. The results suggest that inhibition of TGF-β1 improves pregnancy outcomes by restoring endometrial receptivity in mice with adenomyosis. Similar content being viewed by others

Ferenczy A. Pathophysiology of adenomyosis. Hum Reprod Update. 1998;4(4):312–22. https://doi.org/10.1093/humupd/4.4.312. Leyendecker G, Wildt L, Mall G. The pathophysiology of endometriosis and adenomyosis: tissue injury and repair. Arch Gynecol Obstet. 2009;280(4):529–38. https://doi.org/10.1007/s00404-009-1191-0. Templeman C, Marshall SF, Ursin G, Horn-Ross PL, Clarke CA, Allen M, et al. Adenomyosis and endometriosis in the California teachers study. Fertil Steril. 2008;90(2):415–24. https://doi.org/10.1016/j.fertnstert.2007.06.027. Bergeron C, Amant F, Ferenczy A. Pathology and physiopathology of adenomyosis. Best Pract Res Clin Obstet Gynaecol. 2006;20(4):511–21. https://doi.org/10.1016/j.bpobgyn.2006.01.016. Shen M, Liu X, Zhang H, Guo SW. Transforming growth factor beta1 signaling coincides with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis in mice. Hum Reprod. 2016;31(2):355–69. https://doi.org/10.1093/humrep/dev314. Liu X, Shen M, Qi Q, Zhang H, Guo SW. Corroborating evidence for platelet-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis. Hum Reprod. 2016;31(4):734–49. https://doi.org/10.1093/humrep/dew018. Zhu B, Chen Y, Shen X, Liu X, Guo SW. Anti-platelet therapy holds promises in treating adenomyosis: experimental evidence. Reprod Biol Endocrinol. 2016;14(1):66. https://doi.org/10.1186/s12958-016-0198-1. Chen YJ, Li HY, Huang CH, Twu NF, Yen MS, Wang PH, et al. Oestrogen-induced epithelial-mesenchymal transition of endometrial epithelial cells contributes to the development of adenomyosis. J Pathol. 2010;222(3):261–70. https://doi.org/10.1002/path.2761. Oh SJ, Shin JH, Kim TH, Lee HS, Yoo JY, Ahn JY, et al. β-Catenin activation contributes to the pathogenesis of adenomyosis through epithelial–mesenchymal transition. J Pathol. 2013;231(2):210–22. https://doi.org/10.1002/path.4224. Nieto MA. Epithelial-Mesenchymal transitions in development and disease: old views and new perspectives. Int J Dev Biol. 2009;53(8–10):1541–7. https://doi.org/10.1387/ijdb.072410mn. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420–8. https://doi.org/10.1172/JCI39104. Nieto MA. The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol. 2011;27:347–76. https://doi.org/10.1146/annurev-cellbio-092910-154036. Loboda A, Sobczak M, Jozkowicz A, Dulak J. TGF-beta1/Smads and miR-21 in renal fibrosis and inflammation. Mediat Inflamm. 2016;2016:8319283–12. https://doi.org/10.1155/2016/8319283. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110(3):341–50. https://doi.org/10.1172/JCI15518. Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-beta signaling in fibrosis. Growth Factors. 2011;29(5):196–202. https://doi.org/10.3109/08977194.2011.595714. Inagaki N, Ung L, Otani T, Wilkinson D, Lopata A. Uterine cavity matrix metalloproteinases and cytokines in patients with leiomyoma, adenomyosis or endometrial polyp. Eur J Obstet Gynecol Reprod Biol. 2003;111(2):197–203. https://doi.org/10.1016/s0301-2115(03)00244-6. Jovanović A, Kramer B. The effect of hyperstimulation on transforming growth factor β1 and β2 in the rat uterus: possible consequences for embryo implantation. Fertil Steril. 2010;93(5):1509–17. https://doi.org/10.1016/j.fertnstert.2008.12.092. Struble J, Reid S, Bedaiwy MA. Adenomyosis: a clinical review of a challenging gynecologic condition. J Minim Invasive Gynecol. 2016;23(2):164–85. https://doi.org/10.1016/j.jmig.2015.09.018. de Ziegler D, Pirtea P, Ayoubi JM. Inflammation and uterine fibrosis: the possible role of chronic endometritis. Fertil Steril. 2019;111(5):890–1. https://doi.org/10.1016/j.fertnstert.2019.02.005. Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet. 2006;7(3):185–99. https://doi.org/10.1038/nrg1808. Navot D, Scott RT, Droesch K, Veeck LL, Liu HC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril. 1991;55(1):114–8. https://doi.org/10.1016/s0015-0282(16)54069-2. Yoshinaga K. A sequence of events in the uterus prior to implantation in the mouse. J Assist Reprod Genet. 2013;30(8):1017–22. https://doi.org/10.1007/s10815-013-0093-z. Kijima K, Kasazumi H, Iwai M, Hatayama H, Fujimoto M, Inoue T, et al. Expression of leukemia inhibitory factor in human endometrium and placenta. Biol Reprod. 1994;50:882–7. https://doi.org/10.1095/biolreprod50.4.882. Aghajanova L. Leukemia inhibitory factor and human embryo implantation. Ann N Y Acad Sci. 2004;1034(1):176–83. https://doi.org/10.1196/annals.1335.020. Salleh N, Giribabu N. Leukemia inhibitory factor: roles in embryo implantation and in nonhormonal contraception. ScientificWorldJournal. 2014;2014:201514–0. https://doi.org/10.1155/2014/201514. Cheng J-G, Chen JR, Hernandez L, Alvord WG, Stewart CL. Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation. Proc Natl Acad Sci. 2001;98(15):8680–5. https://doi.org/10.1073/pnas.151180898. Graf U, Casanova EA, Cinelli P. The role of the leukemia inhibitory factor (LIF) - pathway in derivation and maintenance of murine pluripotent stem cells. Genes (Basel). 2011;2(1):280–97. https://doi.org/10.3390/genes2010280. Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart CL. Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci U S A. 1996;93:3115–20. https://doi.org/10.1073/pnas.93.7.3115. Stewart CL, Kaspart P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, et al. Blastocyst implantation depends on maternal expression of leukemia inhibitory factor. Nature. 1992;359:76–9. https://doi.org/10.1038/359076a0. Yen CF, Liao SK, Huang SJ, Tabak S, Arcuri F, Lee CL, et al. Decreased endometrial expression of leukemia inhibitory factor receptor disrupts the STAT3 signaling in adenomyosis during the implantation window. Reprod Sci. 2017;24(8):1176–86. https://doi.org/10.1177/1933719116681515. Tsai HD, Chang CC, Hsieh YY, Lo H-Y. Leukemia inhibitory factor expression in different endometrial locations between fertile and infertile women throughout different menstrual phases. J Assist Reprod Genet. 2000;17(8):415–8. https://doi.org/10.1023/A:1009457016871. Franasiak JM, Holoch KJ, Yuan L, Schammel DP, Young SL, Lessey BA. Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versus alphanubeta3 testing in women with unexplained infertility. Fertil Steril. 2014;101(6):1724–31. https://doi.org/10.1016/j.fertnstert.2014.02.027. Chen XY, Chen J, Wang ZY, Yu XH, Wei BX, Wu XH. Effects of modified Shoutaiwai recipe on integrin beta3 and leukemia-inhibitory factor in endometrium of controlled ovarian hyperstimulation mice during the implantation window. Genet Mol Res. 2015;14(2):2970–7. https://doi.org/10.4238/2015.April.10.6. Ganesh A, Chauhan N, Das S, Chakravarty B, Chaudhury K. Endometrial receptivity markers in infertile women stimulated with letrozole compared with clomiphene citrate and natural cycles. Syst Biol Reprod Med. 2014;60(2):105–11. https://doi.org/10.3109/19396368.2013.862316. Green AR, Styles JA, Parrott EL, Gray D, Edwards RE, Smith AG, et al. Neonatal tamoxifen treatment of mice leads to adenomyosis but not uterine cancer. Exp Toxicol Pathol. 2005;56(4–5):255–63. https://doi.org/10.1016/j.etp.2004.10.001. Dueholm M. Uterine adenomyosis and infertility, review of reproductive outcome after in vitro fertilization and surgery. Acta Obstet Gynecol Scand. 2017;96(6):715–26. https://doi.org/10.1111/aogs.13158. Harada T, Khine YM, Kaponis A, Nikellis T, Decavalas G, Taniguchi F. The impact of adenomyosis on women’s fertility. Obstet Gynecol Surv. 2016;71(9):557–68. https://doi.org/10.1097/OGX.0000000000000346. Kissler S, Hamscho N, Zangos S, Wiegratz I, Schlichter S, Menzel C, et al. Uterotubal transport disorder in adenomyosis and endometriosis—a cause for infertility. BJOG Int J Obstet Gynaecol. 2006;113(8):902–8. https://doi.org/10.1111/j.1471-0528.2006.00970.x. Jiang Y, Jiang R, Cheng X, Zhang Q, Hu Y, Zhang H, et al. Decreased expression of NR4A nuclear receptors in adenomyosis impairs endometrial decidualization. Mol Hum Reprod. 2016;22(9):655–68. https://doi.org/10.1093/molehr/gaw042. Campo S, Campo V, Benagiano G. Infertility and adenomyosis. Obstet Gynecol Int. 2012;2012:1–8. https://doi.org/10.1155/2012/786132. Guo S, Li Z, Yan L, Sun Y, Feng Y. GnRH agonist improves pregnancy outcome in mice with induced adenomyosis by restoring endometrial receptivity. Drug Des Devel Ther. 2018;12:1621–31. https://doi.org/10.2147/DDDT.S162541. Fischer CP, Kayisili U, Taylor HS. HOXA10 expression is decreased in endometrium of women with adenomyosis. Fertil Steril. 2011;95(3):1133–6. Xiao Y, Sun X, Yang X, Zhang J, Xue Q, Cai B, et al. Leukemia inhibitory factor is dysregulated in the endometrium and uterine flushing fluid of patients with adenomyosis during implantation window. Fertil Steril. 2010;94(1):85–9. https://doi.org/10.1016/j.fertnstert.2009.03.012. Younes G, Tulandi T. Effects of adenomyosis on in vitro fertilization treatment outcomes: a meta-analysis. Fertil Steril. 2017;108(3):483–90. e3. https://doi.org/10.1016/j.fertnstert.2017.06.025. Buggio L, Monti E, Gattei U, Dridi D, Vercellini P. Adenomyosis: fertility and obstetric outcome. A comprehensive literature review. Minerva Ginecol. 2018;70(3):295–302. https://doi.org/10.23736/S0026-4784.17.04163-6. Mochimaru A, Aoki S, Oba MS, Kurasawa K, Takahashi T, Hirahara F. Adverse pregnancy outcomes associated with adenomyosis with uterine enlargement. J Obstet Gynaecol Res. 2015;41(4):529–33. Horton J, Sterrenburg M, Lane S, Maheshwari A, Li TC, Cheong Y. Reproductive, obstetric, and perinatal outcomes of women with adenomyosis and endometriosis: a systematic review and meta-analysis. Hum Reprod Update. 2019;25(5):593–633. Kim J-H, Ham S, Lee Y, Suh GY, Lee Y-S. TTC3 contributes to TGF-β1-induced epithelial− mesenchymal transition and myofibroblast differentiation, potentially through SMURF2 ubiquitylation and degradation. Cell Death Dis. 2019;10(2):92. https://doi.org/10.1038/s41419-019-1308-8. Ling J, Cai Z, Jin W, Zhuang X, Kan L, Wang F, et al. Silencing of c-ski augments TGF-b1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells. Cardiol J. 2019;26(1):66–76. https://doi.org/10.5603/CJ.a2018.0009. Kay N, Huang CY, Shiu LY, Yu YC, Chang Y, Suen JL, et al. The effects of anti-TGF-beta1 on epithelial-mesenchymal transition in the pathogenesis of adenomyosis. Reprod Sci. 2020;27:1698–706. https://doi.org/10.1007/s43032-020-00139-0. Shen M, Liu X, Zhang H, Guo S-W. Transforming growth factor β1 signaling coincides with epithelial–mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis in mice. Hum Reprod. 2016;31(2):355–69. https://doi.org/10.1093/humrep/dev314. Hills CE, Squires PE. TGF-beta1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy. Am J Nephrol. 2010;31(1):68–74. https://doi.org/10.1159/000256659. Arribillaga L, Dotor J, Basagoiti M, Riezu-Boj JI, Borras-Cuesta F, Lasarte JJ, et al. Therapeutic effect of a peptide inhibitor of TGF-beta on pulmonary fibrosis. Cytokine. 2011;53(3):327–33. https://doi.org/10.1016/j.cyto.2010.11.019. Grimbizis GF, Mikos T, Tarlatzis B. Uterus-sparing operative treatment for adenomyosis. Fertil Steril. 2014;101(2):472–87 e8. https://doi.org/10.1016/j.fertnstert.2013.10.025. Norwitz ER. Defective implantation and placentation: laying the blueprint for pregnancy complications. Reprod BioMed Online. 2007;14 Spec No 1:101–9. https://doi.org/10.1016/S1472-6483(10)61464-2. Woods L, Perez-Garcia V, Hemberger M. Regulation of placental development and its impact on fetal growth-new insights from mouse models. Front Endocrinol (Lausanne). 2018;9:570. https://doi.org/10.3389/fendo.2018.00570. Mohiyiddeen L, Hardiman A, Fitzgerald C, Hughes E, Mol BW, Johnson N, et al. Tubal flushing for subfertility. Cochrane Database Syst Rev. 2015;5:CD003718. https://doi.org/10.1002/14651858.CD003718.pub4. Mikolajczyk M, Wirstlein P, Skrzypczak J. The impact of leukemia inhibitory factor in uterine flushing on the reproductive potential of infertile women–a prospective study. Am J Reprod Immunol. 2007;58(1):65–74. https://doi.org/10.1111/j.1600-0897.2007.00492.x. Chu B, Zhong L, Dou S, Wang J, Li J, Wang M, et al. miRNA-181 regulates embryo implantation in mice through targeting leukemia inhibitory factor. J Mol Cell Biol. 2015;7(1):12–22. https://doi.org/10.1093/jmcb/mjv006. McCluggage W, Desai V, Manek S. Tamoxifen-associated postmenopausal adenomyosis exhibits stromal fibrosis, glandular dilatation and epithelial metaplasias. Histopathology. 2000;37(4):340–6. https://doi.org/10.1046/j.1365-2559.2000.01001.x. Acknowledgments This work was supported by the MOST research fund, MOST 107-2320-B-650-001 (SJH), and E-Da Hospital Research Fund, grants EDAHT106006 (CYH), EDAHT107022 (CYH), EDAHT108005 (CYH), and EDPJ107056 (SJH). Author information Authors and Affiliations Corresponding authors Ethics declarations The animal studies were conducted under the E-Da Hospital Institutional Animal Care and Use Committee approval (permit number: IACUC-105024). Conflict of Interest The authors declare that they have no conflict of interest. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions About this article Cite this article Kay, N., Huang, CY., Shiu, LY. et al. TGF-β1 Neutralization Improves Pregnancy Outcomes by Restoring Endometrial Receptivity in Mice with Adenomyosis. Reprod. Sci. 28, 877–887 (2021). https://doi.org/10.1007/s43032-020-00308-1 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-020-00308-1