Analysis of cell type-specific adverse effects in ovaries

<p dir="ltr">The global rise in infertility, coupled with an increasing reliance on assisted reproductive technologies (ART), highlights the growing concern over environmental exposures that are associated with negative impacts on fertility. As the ovary's finite pool of oocytes determines female fertility, it is crucial to study the effects of chemicals on ovarian function.</p><p dir="ltr">The overarching aim of this thesis was to identify key molecular targets and processes affected by chemical exposure in human ovaries, with the ultimate goal of generating novel information that could improve future risk assessment. This aim was approached through two complementary strategies: experimental studies and the development of AOPs. DEHP was chosen as a model compound. In the experimental studies, its primary metabolite, MEHP, was used to expose human ovarian tissue and cell cultures in vitro, followed by transcriptomic analyses to identify effects in an unbiased manner. For AOP development, the focus was on DEHP's hypothesized mode of action, androgen signaling, mapping its role in ovarian follicle biology through semi-systematic literature reviews.</p><p dir="ltr">Paper I aimed to identify the effects of MEHP exposure on human ovarian tissue and ovarian cells in culture, utilizing RNA-sequencing and histological assessments. The findings highlighted significant effects on follicle survival and growth in vitro in human ovarian cortical cultures, along with gene expression changes related to cytoskeleton, adherens junctions and Hippo pathway in ovarian cells.</p><p dir="ltr">Paper IV focused on cell type-specific responses, employing single-cell RNA sequencing to investigate the molecular effects of MEHP exposure on different ovarian cell types. This approach confirmed the disruption of cytoskeleton genes observed in Paper I in more cell types, and identified disruption of oxidative phosphorylation genes. Additionally, it revealed reduced cell interactions upon exposure and identified glial cells as the most sensitive cell types to the exposure.</p><p dir="ltr">Paper II aimed to develop a Key Event Relationship (KER) that links androgen receptor antagonism to reduced granulosa cell proliferation, as part of an AOP on androgen antagonism leading to reduced female fertility (AOP345). The KER was developed through a systematic literature review, as it was non-canonical knowledge, and studies were collected and assessed for their quality.</p><p dir="ltr">Paper III was built on Paper II, by continuing the development of the AOP, populating the information of all the included Key Events (KEs) and KERs. A similar systematic approach was followed to connect granulosa proliferation with ovarian cycle irregularities. A weight-of-evidence approach was followed, to inform the confidence in the complete AOP.</p><p dir="ltr">Overall, this thesis provides new insights into the impact of chemical exposures on ovarian function, advancing understanding through both experimental and AOP-based approaches. In Papers I and IV processes including cytoskeleton organization and oxidative phosphorylation are identified through MEHP results, as potential targets for female fertility endpoints. Furthermore, the future perspective of expanding the assessment of chemical effects beyond follicles and onto ovarian cell types like glial cells is presented. Through Papers II and III a pragmatic way of developing KEs, KERs and whole AOPs is demonstrated. AOP345 highlights the importance of follicle counts as a key endpoint in toxicological assessments, an approach currently considered optional in test guidelines. However, as follicle counting is both subjective and time-consuming, these papers identify the possibility of developing granulosa cell proliferation as an alternative.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>E.M. Panagiotou</b>, A. Damdimopoulos, T. Li, E. Moussaud-Lamodière, M. Pedersen, F. Lebre, K. Pettersson, C. Arnelo, K. Papaikonomou, E. Alfaro-Moreno, C. Lindskog, T. Svingen, P. Damdimopoulou. Exposure to the phthalate metabolite MEHP impacts survival and growth of human ovarian follicles in vitro. Toxicology. 505 (2024). <br><a href="https://doi.org/10.1016/j.tox.2024.153815">https://doi.org/10.1016/j.tox.2024.153815<br></a><br></p><p dir="ltr">II. <b>E.M. Panagiotou</b>, M.K. Draskau, T. Li, A. Hirschberg, T. Svingen, P. Damdimopoulou. AOP key event relationship report: Linking decreased androgen receptor activation with decreased granulosa cell proliferation of gonadotropin-independent follicles. Reproductive Toxicology. 112 (2022) 136-147. <a href="https://doi.org/10.1016/j.reprotox.2022.07.004" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.reprotox.2022.07.004</a></p><p dir="ltr">III. <b>E.M. Panagiotou</b>, H.K.L. Johansson, J. Zilliacus, A. Beronius, T. Svingen, P. Damdimopoulou. AOP report: Androgen receptor (AR) antagonism leading to decreased fertility in females via reduced granulosa cell proliferation. [Submitted]</p><p dir="ltr">IV. <b>E.M. Panagiotou</b>*, L. Méar*, E. Moussaud-Lamodière, A. Kamposiora, N. Throumpari, C. Arnelo, K. Pettersson, K. Papaikonomou, C. Lindskog, P. Damdimopoulou. Single-cell insights on the effects of the phthalate metabolite MEHP on the human ovary in vitro. [Manuscript]</p><p dir="ltr">* Equal contribution authors</p>