Impact of Endocrine-Disrupting Chemicals on Female Reproductive Health: In Vitro Studies on Endometrial Stromal Cells and Pluripotent Stem Cell-Derived Germ Cell Models

Environmental exposure to industrial chemicals such as melamine and polyfluoroalkyl sustances (eg. PFOS) has raised concerns due to their potential as Endocrine Disrupting Chemicals (EDCs) affecting female reproductive health. While their effects have been documented in animal models, the cellular and molecular mechanisms underlying their impact on the human endometrium and early germline remain poorly understood. In the first section, this thesis investigated the effects of melamine on the stromal component of the human endometrium. By reproducing the hormonal dynamics of the menstrual cycle, this section aimed to elucidate how melamine interferes with decidualization processes, oxidative balance, and cellular remodeling, potentially compromising endometrial receptivity. In the second section, the thesis examined the effects of perfluorooctane sulfonate (PFOS) on early human germline development using a pluripotent stem cell–based in vitro model of primordial germ cell–like cell (hPGCLC) differentiation. This approach provided a human-relevant platform to study how PFOS exposure at environmentally relevant concentrations affects hPGCLC specification, differentiation efficiency, and embryoid body morphology. For endometrial studies, hESCs were isolated from healthy women (n=7) and cultured under hormone-simulated conditions replicating the proliferative and mid-secretory phases. Cells were exposed to melamine (0.1 mM) during hormonal stimulation. A comprehensive panel of assays was performed, including cytotoxicity tests, ROS and MitoSOX detection, mitochondrial membrane potential assessment, mtDNA copy number determination, DNA fragmentation analysis, immunofluorescence, Western blotting, wound healing assays, and gene expression analysis to evaluate decidualization hallmarks, mitochondrial function, endoplasmic reticulum stress, and hormonal signaling. For germline studies, two hiPSC lines (CTRL14, female; KICO, male) were maintained under defined culture conditions and differentiated into hPGCLCs using a nine-day protocol. Cells were exposed to PFOS at environmentally relevant concentrations (7–700 nM). Analyses included measurement of cell proliferation and Embryoid body (EB) area using ImageJ. Samples were collected and fixed for immunofluorescence staining to evaluate co-expression of key germline markers. EB dissociation and single-cell preparation were followed by FACS to quantify and sort hPGCLCs. Melamine exposure markedly affected human endometrial stromal cell physiology in a hormone-dependent manner. At sublethal concentrations, it induced mitochondrial oxidative stress and endoplasmic reticulum dysfunction, disrupting mitochondrial morphology, connectivity, and function, particularly during the mid-secretory phase. These stress responses were accompanied by transcriptional dysregulation of key differentiation and hormonal markers (BMP2, FGF2, LIFR, IGFBP1, HOXA10, HOXA11, LHCGR), indicating impaired endometrial receptivity. Melamine also disturbed extracellular matrix remodeling by altering the MMP–TIMP balance. Combined with cytoskeletal disorganization and reduced migratory capacity, these findings demonstrate that melamine disrupts the molecular and structural framework required for proper endometrial remodeling and fertility. PFOS exposure did not affect hiPSC proliferation in either the female (CTRL14) or male (KICO) line, confirming the absence of cytotoxic effects under the tested concentrations. However, during early differentiation, PFOS significantly reduced embryoid body size in CTRL14 in a concentration-dependent manner, while KICO EBs remained largely unaffected, revealing a line-specific sensitivity. Despite these morphological changes, immunofluorescence analysis confirmed the successful induction of hPGCLCs in both lines, with maintained expression of key germ cell markers (TFAP2C, SOX17, and OCT4). Flow cytometry further indicated a non-significant but consistent upward trend in hPGCLC proportions following PFOS exposure, particularly in CTRL14, suggesting that PFOS may subtly promote germ cell specification within the EB environment. These findings demonstrate that environmental pollutants can disrupt female reproductive health at multiple levels: melamine alters hormonal responsiveness and induces oxidative stress in the stromal component of the endometrium, while PFOS perturbs early germline development. This work sheds light on the possible molecular mechanisms underlying the disruption of key reproductive processes by EDCs and underscores the importance of understanding their impact to support stricter environmental regulations aimed at protecting future generations.