P-589 Effects of phthalates on the 3D spheroid cell culture as a model for the human ovarian extracellular matrix
Authors: Nikanfar S , Rivas Leonel E  C , Damdimopoulou P , Flaws J  A , Alves Bento E , Andrade Amorim C 
Published: July 2024

Abstract

Study question
Do phthalates affect the ovary by impacting its extracellular matrix (ECM)?

Summary answer
Phthalate mixture (PM) negatively affects the components of ovarian ECM, as demonstrated by spheroids as a model.

What is known already
The widespread production and pervasive use of phthalates as plasticizers in everyday products significantly increases human exposure to these substances. Their documented toxic effects on reproductive and endocrine functions raise substantial public health concerns. Yet, a full understanding of how phthalates affect ovarian functions and especially the ECM structure in females—more exposed to these products than men—remains incomplete. Moreover, there has been limited investigation in identifying a suitable model capable of mimicking the 3D ovarian ECM structure in vitro for studying phthalate impacts.

Study design, size, duration
Stromal cells were isolated from the ovarian cortex of post-menopausal organ donors. Cells were seeded onto agarose micro-well molds to form the spheroids. After 6 days, spheroids were treated with a PM (0 and 100 µg/ml) for 4 days. Cell proliferation, spheroids diameter and protein levels of ECM components were measured for both groups.

Participants/materials, setting, methods
Ovaries from four deceased post-menopausal multi-organ donors (IRB reference 2018/19DEC/475) were used for cell isolation. After seeding of cells, their diameter was measured on days 6 (start of treatment) and 10 (end of treatment) using an inverted microscope to assess their development. The study measured the protein levels of collagen VI, Emilin, and Fibrilin using immunohistochemistry and Ki67 using immunofluorescence in ovarian spheroids. The picrosirius red staining (PSR) was used to measure the collagen content.

Main results and the role of chance
The evaluation of spheroid diameter indicated comparable sizes on day 6 for both groups (0 µg/ml PM: 109±1.3 µm; 100 µg/ml PM: 112.5±1.1 µm). By day 10, there were no notable differences in spheroid size for the group exposed to PM in comparison to the control group (0 µg/ml PM: 104±1.2 µm; 100 µg/ml PM: 107.2±1.2 µm). The assessment of proliferation revealed no significant difference in the expression of ki67-positive cells between the groups (0 µg/ml PM: 3.211±0.64 %; 100 µg/ml PM: 3.379±0.61%; Fig 1).
On the other hand, immunostaining results displayed a decrease in EMILIN-1 (171.6±5.31 vs 201.9±4.71 in control, P &lt; 0.0001) and fibrillin levels (53.1±3.34 vs. 88.3±3.68 in control, P &lt; 0.0001) post-PM treatment. Conversely, collagen VI levels were significantly higher in spheroids treated with PM compared to the control group (122.8±4.99 vs 104.8±4.04, P = 0.005). Although PSR revealed an increase following treatment (9.62±0.75 vs 8.64±0.70 in control), this change did not reach statistical significance (P = 0.344).

Limitations, reasons for caution
This study exclusively focused on ovarian stromal cells from post-menopausal women. Further research is needed to explore the impacts of PM on ECM of prepubertal and reproductive-age women.

Wider implications of the findings
Our study highlights the detrimental impact of phthalates on the ECM of postmenopausal women. Findings suggest ovarian stiffness by decreased elastogenesis-associated proteins (EMILIN-1, fibrillin-1) and increased collagen VI (not I, III) post-phthalate exposure.This pioneering study prompts further exploration of phthalate-induced ECM changes and their potential influence on steroidogenesis and folliculogenesis.

Trial registration number
not applicable