Targeting the ovary : mapping mechanisms to link endocrine-disrupting chemicals to female fertility

Sammanfattning: In recent decades, growing attention has been paid to endocrine-disrupting chemicals (EDCs) as mounting evidence suggests that they can cause detrimental reproductive outcomes in animals and humans. Even though epidemiological studies show the link between EDC exposure and adverse reproductive outcomes such as sub-fertility, longer time-to-pregnancy, and decreased ovarian reserve in women, the underlying mechanisms are not fully elucidated. This is challenging for chemical regulation as we need mechanistic information to properly identify EDCs that may cause detrimental effects. In this thesis, we aim to map the mechanisms linking EDCs to female infertility, identify potential biomarkers of exposure using in vitro culture systems, and carry out cross-species comparisons using rat models. Paper I characterized the effects of in vitro culture alone on ovarian cortical tissue using transcriptomic profiling and investigated the feasibility of using this model to study the effects of exposure to EDCs on the human ovary. RNA sequencing (RNA-seq) revealed a marked change induced by tissue fragmentation and culture itself, including changes in energy metabolism (i.e., glycolysis). Follicles were activated to grow during the culture, which could be explained by the disruption of the Hippo signaling, and partially, by upregulation of the glycolysis pathway. Papers II-III investigated the effects of selected chemicals (pharmaceuticals: DES, KTZ; persistent organic pollutants (POPs): HCB, DDE, PCB156, PCB180, PFOS, and their mixture) on ovarian cells and tissue in culture. All exposures affected follicle growth in culture. Additionally, exposure to POPs resulted in increased follicle atresia in in vitro culture. Using transcriptomic profiling, we found disruption of lipid biology and energy homeostasis as well as increased oxidative stress as potential novel mechanisms connecting chemical exposures to disrupted folliculogenesis. Furthermore, we identified stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) as potential biomarkers of chemical exposure. Papers IV-V investigated the reproductive outcomes induced by DES and KTZ exposure in rats and explored the changes in endpoint sensitivity during pubertal and adult exposure. Moreover, we also developed and assessed the feasibility of using surface photo counting (SPC) as a fast tool to prioritize chemical exposure groups for further histological evaluation. In general, we found that high-dose exposure to DES and KTZ disrupted folliculogenesis in rats. When comparing endpoint sensitivity between different exposure periods, our results suggested that no profound differences can be observed, although pubertal exposure allowed the inclusion of vaginal opening as a sensitive endpoint to estrogenic chemicals. In addition, we showed that the quantification results obtained from the SPC method were significantly correlated with that of traditional histological assessment. Therefore, SPC could be used as a complementary method to prioritize groups for histology analysis. In summary, in vitro ovarian tissue culture can be used to study the impact of chemicals on follicle survival and growth, and underlying mechanisms. Utilizing this model, we found that exposure to the selected chemicals affected folliculogenesis, through disruption of their energy metabolism and increased oxidative stress. Similarly, we showed that high-dose DES and KTZ exposure disrupted follicle growth in rats, but not in low- and middle-dose groups. This suggests that the investigated endpoints in the in vivo study were not sensitive enough. It advocates the need for a sensitive and human-relevant assay for the screening of EDCs present in the global market. The identified common signatures and biomarkers might be used as a base for the future development of such screening assays after validation. Even though this is a small step, we are moving towards the future of a chemical-safe world.