Deciphering the negative impacts of uterine fibroids on endometrial receptivity using 3d organoid system; molecular mechanisms and potential therapeutics

Uterine fibroids (UFs) are the most common benign tumor in reproductive age women. UFs negatively impair endometrial receptivity (ER). Yet, mechanism isn’t fully clear. ER is influenced by hormones, cytokines, growth factors, microRNAs as well as immune cells, mainly natural killer (NK) cells, which plays key role in implantation. Our objective is to explore UF effects on ER using 3D organoid system. Stem cells isolated from UFs or normal myometrium (MyoN) tissues were used to develop 3D organoids. RNA-seq was employed to comparatively profile their transcriptomes. Organoids were stained for inflammatory markers TNF-α and NF-kB (p65) by immunohistochemistry (IHC). Organoids secretome was analyzed for 48-cytokine array as well as TGF-β1 using multiplex ELISA. Exosomal miRNA was extracted from organoids secretome and differential gene expression of miR223-3p, 494-3p and 150 was measured. UFs organoid secretome was exposed to (1) 3D endometrial organoids established from primary epithelial and stromal cells and then ER related markers insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL) were measured using qRT-PCR. (2) cultured CD56+ CD16- NK cells and cell proliferation was measured using XTT assay after 24hr. Also, NK cells were treated with TGF-b1 (10ng/ml) or its receptor inhibitor LY-364947 (2μM) and viability was measured. UFs organoids were treated with VitaminD3 (VitD3 100nM) or green tea extract (EGCG 100μM) for 48hr and IHC, exosomal miRNA expression, cytokine array and effect on NK cell viability was explored using same markers. Unpaired student t-test was used for statistical significance detection. RNA-seq analysis showed enhanced inflammatory signaling in UFs organoids compared to MM using DisGeNET (adjp=0.00001, gene ratio 40/407). UF organoids secreted higher levels of many cytokines including IL-6, IL-8, and TNF-α than MyoN organoid and showed higher expression of TNF-α, and p-NF-kB using IHC (p<0.05). UF organoids secreted higher exosomal miR-223-3p, miR-494-3p, and miR-150 vs. MyoN (p<0.05) which are associated with recurrent implantation failure, infertility and impaired ER. Importantly, UF organoid secretome downregulated IGFBP1 and PRL expression in endometrial organoids compared to untreated control (p<0.05). UFs organoids secreted more TGF-b1 compared to MyoN Which might justify NK cells growth inhibition following UF organoid secretome, compared to MyoN, since TGF-β1 treatment inhibited NK cell growth while its inhibitor restored it (p<0.05). Treating UFs organoids with VitD3 or EGCG induced reduction of IL-6, IL-8, TNF-α and miRs (223-3p, 494-3p and 150) secretion. Moreover, it restored NK cells viability compared to untreated UFs secretome (p<0.05). UFs may negatively impact ER directly through secretion of cytokines and miRNA that interfere with implantation and indirectly via disrupting NK cells viability. VitD3/EGCG might offer beneficial effects by interrupting such negative crosstalk.