The Effects of Di (2-ethylhexyl) Phthalate (DEHP) on Ovarian Endometriotic Spheroids Using a Gelatin Hydrogel Model

Sarah Ibrahim^1,6, Hannah Theriault^2,6, Kathryn B.H. Clancy^4,6, Brendan A.C. Harley^2,3,5,6, Romana A. Nowak^1,6.

¹Department of Animal Sciences; ²Department of Bioengineering; ³Department of Chemical and Biomolecular Engineering; ⁴Department of Anthropology; ⁵Cancer Center at Illinois; ⁶Carl R. Woese Institute for Genomic Biology at the University of Illinois Urbana-Champaign (UIUC), Urbana, USA


Abstract Text:

Phthalates are widely used plasticizers found in many consumer products, leading to significant human exposure. As endocrine-disrupting chemicals, they can interfere with hormone function, particularly impacting the reproductive system. Their ability to mimic or block hormones raises concerns about potential health effects. Endometriosis is a major reproductive health disease impacting 1 out of 10 cycling women of childbearing age. It is characterized by endometrial tissue outside the uterus, commonly on the ovary, peritoneum, and other pelvic organs. Estradiol is considered a key hormone for the growth and progression of endometriotic lesions and promoting inflammation. Di (2- ethylhexyl) phthalate (DEHP) is a widely used phthalate and has been shown to have estrogenic and proinflammatory effects. DEHP may play a role in endometriosis lesion progression through one of these pathways. However, the mechanisms involved, and the underlying cause have not been well studied. Previous in vitro work has examined the impact of DEHP on healthy endometrial cells and cancer-derived endometrial cells. Here we describe a 3D tissue engineering model to encapsulate endometriotic spheroids in physiologically relevant extracellular matrix to investigate the effects of DEHP on cell invasion, a critical early step in endometriotic lesion initiation. We created spheroids using cohorts of epithelial and stromal cells embedded into a 3D methacrylamide-functionalized gelatin (GelMA) hydrogel. Endometriotic cells were azurite-blue tagged human endometrial-endometriotic stromal cells (azb-iEC-ESCs; Fazleabas Lab, Michigan State University) and GFP-transduced human endometriotic epithelial cells (12z; ABM). Spheroids were formed from 5000 cells (1:3 mixture of GFP-12z: azb-iEC-ESCs), encapsulated in a GelMA precursor suspension, then UV polymerized. GelMA was synthesized utilizing a previously established method by the Harley Lab. GelMA was characterized with NMR (Agilent 600MHz NMR), and compressive testing (Instron 5943). 3D hydrogels were prepared with 5 wt% or 7 wt% GelMA, PBS, and 0.1% w/v lithium phenyl phosphinate photoinitiator. GelMA was photopolymerized under a UV light (λ = 365 nm, 7.14 mW cm–2, AccuCure Spot System ULM-3-365) for 30s. Spheroids were cultured in a 50/50 mixture of 12z complete culture medium (CCM) and iEC-ESC CCM. Media (0 μM DEHP, 1 μM DEHP, 0.1 μM DEHP, or 0.01 μM DEHP treatments) were changed every two days and spheroids were imaged on D0, D3, D6, D9, and D14. GelMA synthesized at 5 wt% and UV polymerized for 30 seconds fell within the stiffness of normal, healthy ovarian tissue (3.3 ± 2.5 kPa), with 7 wt% hydrogels above this desired stiffness. We tested environmentally relevant doses of DEHP and observed differences in cell outgrowth patterns, with the 1μM DEHP-treated spheroids experiencing significantly more outgrowth by D3. We observed visual differences in the spheroid outgrowth of control vs. DEHP treatments. Transcriptomic analysis revealed that the mRNA levels of cytokines, including IL6, IL8, and VEGF, were trending higher in the treatment groups at D8 in comparison to D1. Surprisingly, the expression of IL1-b was significantly reduced by D8 at the lowest dose of DEHP. Ongoing studies are examining differences in the expression of inflammatory cytokines at D14 as well as potential differences in proliferation of the cells in these spheroids. Our results show that this 3-D spheroid culture system can be used to directly test effects of environmental chemicals on processes involved in initiation and progression of endometriomas. (Funded by R01 ES034112 to RAN, R01 CA256481 to BACH and IETP program)