Forskolin versus cAMP-Induced Decidualization and Survival of Endometrial Stromal Cells of Endometriosis Patients

Impairment of decidualization of eutopic human endometrial stromal cells (hESCs) may cause an increase in cell survival of endometrial tissue in the peritoneal cavity constituting a precondition for endometriosis development. Decidualization is a physiological process involving progesterone action and cAMP signaling. We here evaluated the effect of 8-Br-cAMP, the adenylate cyclase activator forskolin and of the progestin progesterone and medroxyprogesterone acetate (MPA) alone and in combination on decidualization induction using prolactin ELISA, and on cell size, cell granularity, and cell survival via flow cytometry in hESCs of patients with and without endometriosis. While progestins alone did not induce functional decidualization in hESCs, 8-Br-cAMP and forskolin induced decidualization in hESCs from both cohorts, whereas the induction of FOXO1 transcription and prolactin secretion by forskolin was significantly lower than by 8-Br-cAMP. 8-Br-cAMP- and forskolin-induced prolactin secretion was significantly enhanced by MPA, but not by progesterone. Decidualization entailed a decrease in cell size and in cell granularity. In general, hESCs from women with mild (ASRM I/II) as well as severe (ASRM III/IV) endometriosis in trend displayed a higher granularity, whereas mainly hESCs from severe endometriosis showed a stronger resistance to the induction of cell death after decidualization induction. In both cohorts, the amount of the decidual marker protein prolactin rather exhibited an anti-proportional correlation to cell death induction during six day treatment. This study contributes to widen our understanding of the connection of decidualization and cell death in endometriosis. Similar content being viewed by others Data Availability The data underlying this article will be shared on reasonable request to the corresponding author. Code Availability This is not applicable.

Giudice LC. Endometriosis. N Engl J Med. 2010;362:2389–98. https://doi.org/10.1056/NEJMcp1000274. Simoens S, Dunselman G, Dirksen C, Hummelshoj L, Bokor A, Brandes I, Brodszky V, Canis M, Colombo GL, DeLeire T, Falcone T, Graham B, Halis G, Horne A, Kanj O, Kjer JJ, Kristensen J, Lebovic D, Mueller M, et al. The burden of endometriosis: costs and quality of life of women with endometriosis and treated in referral centres. Hum Reprod. 2012;27:1292–9. https://doi.org/10.1093/humrep/des073. Meuleman C, Vandenabeele B, Fieuws S, Spiessens C, Timmerman D, D'Hooghe T. High prevalence of endometriosis in infertile women with normal ovulation and normospermic partners. Fertil Steril. 2009;92:68–74. https://doi.org/10.1016/j.fertnstert.2008.04.056. Sampson JA. Metastatic or embolic endometriosis, due to the menstrual dissemination of endometrial tissue into the venous circulation. Am J Pathol. 1927;3:93–110.43. Burney RO, Giudice LC. 10.1. The pathogenesis of endometriosis. In: Nezhat C, Nezhat F, Nezhat C, editors. Nezhat's operative gynecologic laparoscopy with hysteroscopy. Cambridge: Cambridge University Press; 2008. p. 251–7. Nisolle M, Casanas-Roux F, Donnez J. Early-stage endometriosis: adhesion and growth of human menstrual endometrium in nude mice. Fertil Steril. 2000;74:306–12. https://doi.org/10.1016/s0015-0282(00)00601-4. Gellersen B, Brosens JJ. Cyclic decidualization of the human endometrium in reproductive health and failure. Endocr Rev. 2014;35:851–905. https://doi.org/10.1210/er.2014-1045. Tang B, Gurpide E. Direct effect of gonadotropins on decidualization of human endometrial stroma cells. J Steroid Biochem Mol Biol. 1993;47:115–21. https://doi.org/10.1016/0960-0760(93)90064-4. Gellersen B, Brosens IA, Brosens JJ. Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med. 2007;25:445–53. https://doi.org/10.1055/s-2007-991042. Bulun SE, Cheng YH, Yin P, Imir G, Utsunomiya H, Attar E, Innes J, Julie KJ. Progesterone resistance in endometriosis: link to failure to metabolize estradiol. Mol Cell Endocrinol. 2006;248:94–103. https://doi.org/10.1016/j.mce.2005.11.041. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA, Giudice LC. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology. 2007;148:3814–26. https://doi.org/10.1210/en.2006-1692. Patel BG, Rudnicki M, Yu J, Shu Y, Taylor RN. Progesterone resistance in endometriosis: origins, consequences and interventions. Acta Obstet Gynecol Scand. 2017;96:623–32. https://doi.org/10.1111/aogs.13156. Klemmt PA, Carver JG, Kennedy SH, Koninckx PR, Mardon HJ. Stromal cells from endometriotic lesions and endometrium from women with endometriosis have reduced decidualization capacity. Fertil Steril. 2006;85:564–72. https://doi.org/10.1016/j.fertnstert.2005.08.046. Lessey BA, Lebovic DI, Taylor RN. Eutopic endometrium in women with endometriosis: ground zero for the study of implantation defects. Semin Reprod Med. 2013;31:109–24. https://doi.org/10.1055/s-0032-1333476. Kokawa K, Shikone T, Nakano R. Apoptosis in the human uterine endometrium during the menstrual cycle. J Clin Endocrinol Metab. 1996;81:4144–7. https://doi.org/10.1210/jcem.81.11.8923873. Boeddeker SJ, Hess AP. The role of apoptosis in human embryo implantation. J Reprod Immunol. 2015;108:114–22. https://doi.org/10.1016/j.jri.2015.02.002. Mikhailov V. Life cycle of decidual cells. In: Jeon KW, editor. International review of cytology. San Diego: Academic Press; 2003. p. 1–63. Dmowski W, Ding J, Shen J, Rana N, Fernandez B, Braun D. Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod. 2001;16:1802–8. https://doi.org/10.1093/humrep/16.9.1802. Gebel HM, Braun DP, Tambur A, Frame D, Rana N, Dmowski WP. Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis. Fertil Steril. 1998;69:1042–7. https://doi.org/10.1016/s0015-0282(98)00073-9. Gellersen B, Brosens J. Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol. 2003;178:357–72. https://doi.org/10.1677/joe.0.1780357. Singer BB, Klaile E, Scheffrahn I, Müller MM, Kammerer R, Reutter W, Öbrink B, Lucka L. CEACAM1 (CD66a) mediates delay of spontaneous and Fas ligand-induced apoptosis in granulocytes. Eur J Immunol. 2005;35:1949–59. https://doi.org/10.1002/eji.200425691. Kajihara T, Tanaka K, Oguro T, Tochigi H, Prechapanich J, Uchino S, Itakura A, Sucurovic S, Murakami K, Brosens JJ, Ishihara O. Androgens modulate the morphological characteristics of human endometrial stromal cells decidualized in vitro. Reprod Sci. 2014;21:372–80. https://doi.org/10.1177/1933719113497280. Edwards R. Physiological and molecular aspects of human implantation. Hum Reprod. 1995;10:1–13. https://doi.org/10.1093/humrep/10.suppl_2.1. Psychoyos A. Uterine receptivity for nidation. Ann N Y Acad Sci. 1986;476:36–42. https://doi.org/10.1111/j.1749-6632.1986.tb20920.x. Brar AK, Frank GR, Kessler CA, Cedars MI, Handwerger S. Progesterone-dependent decidualization of the human endometrium is mediated by cAMP. Endocrine. 1997;6:301–7. https://doi.org/10.1007/BF02820507. Brosens JJ, Hayashi N, White JO. Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells. Endocrinology. 1999;140:4809–20. https://doi.org/10.1210/endo.140.10.7070. Tseng L, Gao JG, Chen R, Zhu HH, Mazella J, Powell DR. Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells. Biol Reprod. 1992;47:441–50. https://doi.org/10.1095/biolreprod47.3.441. Kusama K, Yoshie M, Tamura K, Imakawa K, Isaka K, Tachikawa E. Regulatory action of calcium ion on cyclic AMP-enhanced expression of implantation-related factors in human endometrial cells. PLoS One. 2015;10:e0132017. https://doi.org/10.1371/journal.pone.0132017. Koch Y, Wimberger P, Grummer R. Human chorionic gonadotropin induces decidualization of ectopic human endometrium more effectively than forskolin in an in-vivo endometriosis model. Exp Biol Med (Maywood). 2018;243:953–62. https://doi.org/10.1177/1535370218782658. Schwede F, Maronde E, Genieser H-G, Jastorff B. Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol Ther. 2000;87:199–226. https://doi.org/10.1016/S0163-7258(00)00051-6. Stork PJ, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12:258–66. https://doi.org/10.1016/S0962-8924(02)02294-8. Yamamizu K, Yamashita JK. Roles of cyclic adenosine monophosphate signaling in endothelial cell differentiation and arterial-venous specification during vascular development. Circ J. 2011;75:253–60. https://doi.org/10.1253/circj.CJ-10-0915. Alberini CM, Ghirardi M, Huang YY, Nguyen PV, Kandel ER. A molecular switch for the consolidation of long-term memory: cAMP-inducible gene expression. Ann N Y Acad Sci. 1995;758:261–86. https://doi.org/10.1111/j.1749-6632.1995.tb24833.x. Aronica SM, Kraus WL, Katzenellenbogen BS. Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci USA. 1994;91:8517–21. https://doi.org/10.1073/pnas.91.18.8517. Tanaka N, Miyazaki K, Tashiro H, Mizutani H, Okamura H. Changes in adenylyl cyclase activity in human endometrium during the menstrual cycle and in human decidua during pregnancy. J Reprod Fertil. 1993;98:33–9. https://doi.org/10.1530/jrf.0.0980033. Hanoune J, Defer N. Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol. 2001;41:145–74. https://doi.org/10.1146/annurev.pharmtox.41.1.14. Laurenza A, Sutkowski EM, Seamon KB. Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action? Trends Pharmacol Sci. 1989;10:442–7. https://doi.org/10.1016/S0165-6147(89)80008-2. Seamon K, Daly J. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J Cyclic Nucleotide Res. 1981;7:201–24. Aghajanova L, Horcajadas JA, Weeks JL, Esteban FJ, Nezhat CN, Conti M, Giudice LC. The protein kinase A pathway-regulated transcriptome of endometrial stromal fibroblasts reveals compromised differentiation and persistent proliferative potential in endometriosis. Endocrinology. 2010;151:1341–55. https://doi.org/10.1210/en.2009-0923. Bruner-Tran KL, Zhang Z, Eisenberg E, Winneker RC, Osteen KG. Down-regulation of endometrial matrix metalloproteinase-3 and -7 expression in vitro and therapeutic regression of experimental endometriosis in vivo by a novel nonsteroidal progesterone receptor agonist, tanaproget. J Clin Endocrinol Metab. 2006;91:1554–60. https://doi.org/10.1210/jc.2005-2024. Moghissi KS, Boyce CR. Management of endometriosis with oral medroxyprogesterone acetate. Obstet Gynecol. 1976;47:265–7. Leno-Duran E, Ruiz-Magana MJ, Munoz-Fernandez R, Requena F, Olivares EG, Ruiz-Ruiz C. Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner. Hum Reprod. 2014;29:2269–77. https://doi.org/10.1093/humrep/deu202. Wu WX, Brooks J, Glasier AF, McNeilly AS. The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy. J Mol Endocrinol. 1995;14:255–61. https://doi.org/10.1677/jme.0.0140255. Wynn RM. Ultrastructural development of the human decidua. Am J Obstet Gynecol. 1974;118:652–70. https://doi.org/10.1016/s0002-9378(16)33740-1. Monice FL, Andrade CG, Abrahamsohn PA, Soto-Suazo M, Oliveira SF, Zorn TM. Granulated decidual cells in the mouse deciduoma: a putative source of decidual prolactin in mice. Cells Tissues Organs. 2001;168:252–63. https://doi.org/10.1159/000047842. Meresman GF, Vighi S, Buquet RA, Contreras-Ortiz O, Tesone M, Rumi LS. Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis. Fertil Steril. 2000;74:760–6. https://doi.org/10.1016/s0015-0282(00)01522-3. Taniguchi F, Kaponis A, Izawa M, Kiyama T, Deura I, Ito M, Iwabe T, Adonakis G, Terakawa N, Harada T. Apoptosis and endometriosis. Front Biosci (Elite Ed). 2011;3:648–62. https://doi.org/10.2741/e277. Vaskivuo T, Stenbäck F, Karhumaa P, Risteli J, Dunkel L, Tapanainen JS. Apoptosis and apoptosis-related proteins in human endometrium. Mol Cell Endocrinol. 2000;165:75–83. https://doi.org/10.1016/S0303-7207(00)00261-6. Sweeney EE, Fan P, Jordan VC. Molecular modulation of estrogen-induced apoptosis by synthetic progestins in hormone replacement therapy: an insight into the women’s health initiative study. Cancer Res. 2014;74:7060–8. https://doi.org/10.1158/0008-5472.Can-14-1784. Acknowledgments The authors thank Hildegard Lax for support in statistical calculation. Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant number GR 1138/14-1, BI 1963/6-1). Author information Authors and Affiliations Contributions All authors participated in the design, interpretation of the studies and analysis of the data, and review of the manuscript. FP and JM conducted the experiments. APB and DBB supplied human endometrial samples. RG and APB created the concept. RG, APB, and BBS developed the methodology. FP and BBS validated the data. FP, JM, and RG wrote the manuscript. GW, BBS, and APB reviewed and edited the manuscript. Corresponding author Ethics declarations Ethical Approval The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the University of Düsseldorf (study number 5535, date of approval 24/05/2016). Consent to Participate This is not applicable. Consent for Publication This is not applicable. Conflict of Interest The authors declare no competing interests. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Platt, F., Moyer, J., Singer, B.B. et al. Forskolin versus cAMP-Induced Decidualization and Survival of Endometrial Stromal Cells of Endometriosis Patients. Reprod. Sci. 30, 2680–2691 (2023). https://doi.org/10.1007/s43032-023-01235-7 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-023-01235-7