(P-509) Molecular Mechanisms Underlying the Adverse Outcomes of DEHP Exposure on Human Preovulatory Follicles

Abstract Authors: Agne Velthut-Meikas¹, Inge Varik¹, Andrea Bellavia ², Runyu Zou ³, Richelle D. Björvang⁴, Kristine Rosenberg¹, Ylva Sjunnesson⁵, Ida Hallberg⁵, Jan Holte⁶, Anne Pikki⁶, Virissa Lenters³, Lützen Portengen³, Jacco Koekkoek⁷, Marja Lamoree⁷, Majorie Van Duursen⁷, Mikael Pedersen⁸, Terje Svingen⁸, Roel Vermeulen³, Andres Salumets^4,9, Pauliina Damdimopoulou⁴

1. Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia;

2. Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA;

3. Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands;

4. Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden;

5. Department of Clinical Sciences, Division of Reproduction, The Center for Reproductive Biology in Uppsala, Swedish University of Agricultural Sciences, Uppsala, Sweden;

6. Department of Women´s and Children´s Health, Uppsala University, Uppsala, Sweden;

7. Amsterdam Institute for Life and Environment, Section Environment and Health, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands;

8. National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark;

9. Competence Center on Health Technologies, Tartu, Estonia;

Abstract Text: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer known to possess endocrine disrupting activities. DEHP may enter the human body through leaching from everyday plastic objects. It is extensively metabolised upon uptake forming a range of hydrolysed and oxidized metabolites that are considered the active endocrine disrupting forms. While DEHP metabolites have been extensively investigated in relation to male reproductive disorders, rodent studies have also raised concern that DEHP may affect ovarian folliculogenesis and steroid hormone production, ultimately affecting fertility in females. In a previous study, we reported a significant association between DEHP metabolites in the human follicular fluid (FF) and reduced responsiveness to controlled ovarian stimulation during IVF treatments. Here, we investigated the mechanisms underlying these effects though molecular analysis of follicular fluid samples.

To explore the molecular cues altered by DEHP exposure in humans, samples from 96 IVF patients from Swedish (N=48) and Estonian (N=48) infertility clinics were analysed. The patients were stratified into high (mean 7.7 ± SD 2.3 nM, N=48) and low (0.8 ± 0.4 nM, N=48) DEHP exposure groups according to the molar sum of the measured levels of 4 DEHP metabolites MEHP, MEHHP, MEOHP and MECPP in their FF samples. Extracellular miRNA levels and 15 steroid hormone concentrations were measured from the same samples by small RNA sequencing and LC-MS/MS, respectively. In addition, FF somatic cell samples from the individual follicles were available in the Estonian cohort, and these were used to measure mRNA expression levels by RNA-seq.

The results were compared between the high and low DEHP groups using the cohort as a covariate. In addition, data integration was used to understand the interactions between the disturbed miRNA, mRNA and steroid levels within a single follicle.

Differential expression (DE) and miRNA:mRNA network analysis revealed that the expression levels of the majority of genes in the cholesterol biosynthesis and steroidogenesis pathways were significantly decreased (FDR< 0.05). The DE miRNAs (FDR< 0.1) were predicted to target a considerable number of key enzymes in these pathways (FDR< 0.05). The down-regulation of CYP17A1 (log₂FC=-2.34, FDR=0.022) in the somatic cells was linked to a reduced progesterone to 17-OH-progesterone ratio (p=0.046) in the FF of women in the high DEHP group. The expression levels of genes involved in inflammatory processes were significantly elevated in the women from the high DEHP group (FDR< 0.05). Following a computational cell-type deconvolution approach, an indication for the infiltration of cells expressing leukocyte markers of M1 and M2 macrophages, T-cells (p< 0.01), as well as neutrophils (p< 0.05) was observed.

In conclusion, exposure to DEHP metabolites significantly alters the follicular milieu within human ovaries: the inflammatory environment is stimulated, while lipid and steroid synthesis are obstructed. This study contributes to understanding the molecular mechanisms by which DEHP exposure may disrupt ovarian sensitivity to pituitary hormones in humans.