In vitro investigation of the teratogenic effects of muscle relaxant antagonists, acetylcholinesterase inhibitors, and Sugammadex on rat embryos

Objective: This study aims to investigate the safety profiles and potential teratogenic effects of neostigmine—an acetylcholinesterase inhibitor—and sugammadex in an in vitro setting by evaluating both organ rudiments and cellular responses using flow cytometry. Methods: In the study, 9.5-day-old rat embryos were explanted and cultured in vitro for 48 hours in rat serum. While full rat serum was used as the culture medium for the control group, the experimental groups received rat serum supplemented with sugammadex at concentrations of 50, 100, 150, and 200 µg/mL, and neostigmine at concentrations of 4, 7, 10, and 13 µg/mL. The dose-dependent effects of these agents on embryonic developmental parameters, as well as their morphological and cellular impacts, were compared using flow cytometry. Each embryo was evaluated for the presence of any malformations. Results: In the groups treated with sugammadex, the morphological score was significantly reduced only at the highest dose of 200 µg/mL (p < 0.05), with no changes observed in the other parameters. At the cellular level, a marked increase in early apoptosis rates was detected starting from 100 µg/mL, while significant increases in late apoptosis and necrosis rates were observed at 200 µg/mL. In the neostigmine groups, morphological parameters were found to be similar to the control (p > 0.05); however, significant increases in both early and late apoptosis rates were observed starting from 4 µg/mL, and in necrosis rates from 7 µg/mL onwards. Conclusion: At a high dose of 200 µg/mL, sugammadex caused damage at both the morphological and cellular levels, whereas neostigmine increased apoptosis and necrosis at the cellular level starting from low doses. The findings indicate that dosage is of critical importance when using these agents during pregnancy. Similar in vitro studies reported in the literature have also shown that morphological deterioration does not always coincide with cellular-level responses, highlighting the importance of conducting advanced cellular analyses in addition to morphological evaluations. To the best of our knowledge, this is the first study to evaluate teratogenic effects through both the examination of organ primordia and the assessment of cellular responses using flow cytometry.