Proteomic profiling reveals the molecular signatures of chemotherapy-induced human ovarian damage.

study questionHow does first-line chemotherapy alter follicular survival and the ovarian microenvironment? SUMMARY ANSWER: First-line chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) induces follicular DNA damage and apoptosis, and causes microenvironmental alterations including immune dysfunction, increased hypoxia and apoptosis, impaired cell cycle and DNA repair capacity, and disruption of the extracellular matrix (ECM). WHAT IS KNOWN ALREADY: Although the mechanisms underlying chemotherapy-induced damage to germ cells are being increasingly deciphered, its impact on the ovarian microenvironment remains largely unexplored. The ovarian stroma is equally exposed to chemotherapy, and since its cells and components are in active communication with follicles, any microenvironmental changes induced by chemotherapy might affect follicles as well. STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian cortex samples from 10 cancer patients (aged 24-30 years) who donated tissue for research purposes were analyzed. Of the 10 patients, 5 had received first-regimen chemotherapy and 5 age-matched controls had not undergone chemotherapy prior to OTC. Chemotherapy-induced ovarian injury was evaluated by comparing stromal and follicular alterations between chemotherapy-exposed and control patients. PARTICIPANTS/MATERIALS, SETTING,

methodsCryopreserved ovarian cortex were processed directly after thawing. Proteins and biological processes dysregulated in response to chemotherapy were identified by mass spectrometry, and the data obtained were validated by western blotting and immunohistochemistry. Stromal and follicular alterations were further examined by (immuno)histochemistry, focusing on apoptosis (TUNEL), DNA damage (γH2Ax), oxidative stress (8-OHdG), proliferation (Ki67), fibrosis (picrosirius red), and analysis of follicle number, developmental stage and morphology. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 5209 proteins were detected in both chemotherapy-exposed and control ovaries, of which 237 proteins (4.5%) showed differential expression. Biological pathways related to immune response, hypoxia and apoptosis were upregulated after chemotherapy exposure, while those involved in cell cycle and DNA repair were downregulated. Markers of the ECM network were also dysregulated. Western blotting and immunostaining confirmed the significant upregulation of complement C3 (innate immunity; P = 0.032), SELENBP1 (hypoxia; P = 0.030) and KRT18 (apoptosis; P = 0.015) as well as a non-significant increase in SERPIN A3 level (ECM; P = 0.077) following chemotherapy-exposure, while NCBP2 (DNA repair) was reduced, though not significantly (P = 0.067). Targeted analyses demonstrated that despite increased stromal cell density following chemotherapy treatment (1.84 ± 0.15 × 106 cells/mm3 vs. 1.62 ± 0.13 × 106 cells/mm3; P = 0.036), no fibrosis was observed. Stromal apoptosis and oxidative stress levels were comparable in both groups (P = 0.464 and P = 0.247, respectively). In contrast, first-regimen chemotherapy persistently affected germ cells, increasing follicular apoptosis (P = 0.013), DNA damage (P = 0.033) and possibly morphological defects (P = 0.061), without depleting the ovarian reserve. LIMITATIONS, REASONS FOR CAUTION: Patients previously exposed to chemotherapy had received different low-gonadotoxic chemotherapy regimens at different times before OTC, making it challenging to isolate the effects of individual agents or to differentiate between short- and long-term dysregulated biological processes. Additionally, analyses were performed on a small cohort and results should be interpreted cautiously. WIDER IMPLICATIONS OF THE

findingsThese findings provide evidence that chemotherapy significantly alters both the ovarian germ cells and stroma, emphasizing the need to further investigate the underlying molecular mechanisms and their impact on ovarian function and fertility preservation. They also suggest target proteins which may drive these chemotherapy-associated ovarian damage for future investigations. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by a grant from the Fond National de la Recherche Scientifique de Belgique-FNRS (grant 1.B.218.24F awarded to J.G.). The support is provided by the Funds Suzanne Duchesne, Serge Rousseau and Docteur Jean Gérard, managed by the King Baudouin Foundation, and by the Jaumotte-Demoulin Foundation. The CMMI is supported by the European Regional Development Fund and the Walloon Region. J.G. and I.D. are supported by FNRS as a postdoctoral researcher and a senior research associate, respectively. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.