P-664 Carbendazim in human follicular fluid : first presence and impact in human granulosa cells

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Abstract Study question Are human ovarian cells exposed to carbemdazin and what are the consequences on their viability, proliferation and steroid production? Summary answer Carbendazim is detected in human follicular fluid mainly in PCOS patients and it increases steroid secretion and decreases cell proliferation in primary human granulosa cells. What is known already Carbendazim (Methyl 2-benzimidazolecarbamate) is a fungicide which was banned last year by the european union but is still present in the french groundwaters and soils. It exhibits antimitotic activity in fungal and mammalian cells. Peri-fertilization exposure to carbendazim induces infertility and early pregnancy loss in female hamsters and in male rat, carbendazim has adverse effects on spermatogenesis, also resulting in a reduced fertility. However, no study until now has been performed experiments in human ovarian fluid and cells. Study design, size, duration A single centre cohort study involving 62 patients was performed from 2021-2023 for the analysis of follicular fluid. The effect of carbendazim on human granulosa cells has been determined on granulosa cells from 30 patients with male factor infertility and in the human granulosa cell line, KGN from 2024 and still ongoing. From each patient, only one cycle is included in the study. Participants/materials, setting, methods Sixty-two patients including 16 PCOS was used from 2021-2023 for the follicular fluid analysis and 30 patients with male factor infertility for cell culture. The study was carried out in accordance with the Declaration of Helsinki principles and free informed consent was obtained from all participants. Carbendazim concentrations were assessed by mass spectrophotometry, while steroid concentrations were measured by ELISA assays. Cell viability and proliferation was dertermined by using CCK8 and BrDU assays, respectively. Main results and the role of chance Carbendazim (CBZ) was detected in follicular fluid in PCOS patients (9 out 16), with a maximum concentration of 76 ng/mL but not in patients with male factor infertility. We next investigated the impact of CBZ on the cell viability, proliferation, and steroidogenesis, in both granulosa tumor cell line (KGN) and primary human granulosa cells. Using a CBZ concentration range from 0 to 1 µg/mL, including the highest CBZ dose found in follicular fluid, we demonstrated that CBZ does not affect cell viability whereas it reduced cell proliferation from the concentrationsof 10 ng/mL in KGN cells. At the opposite, we showed that CBZ increased in a concentration dependent manner progesterone and oestradiol secretion from 1 ng/ml in both KGN and primary granulosa cells. Furthermore, in these cells, CBZ (100 ng/ml) significantly improved IGF-1-induced steroid secretion whereas it did not affect FSH or LH effect. The CBZ effect on the mRNA expression of the cholesterol carrier, StAR and the steroidogenesis enzymes and on the oxidative stress is in progress. In addition, we next compare PCOS and normal granulosa cell sensitivity to CBZ. Taken together, carbendazim exposure could alter human folliculogenesis and could contribute to explain some ovarian disorders. Limitations, reasons for caution The number of PCOS and normal patients is limited. For primary granulosa cell culture, cells are from different diameter of follicles. Wider implications of the findings Environmental pollution is more and more important and human infertility with or without known reasons increases. The use of some phytochemical products could explain some ovarian disorders in women. Trial registration number No

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