Methylene blue but not indigo carmine is toxic to human luteal cells in vitro.

Methylene blue (MB) is reported to be teratogenic when injected intra-amniotically. Indigo carmine (IC) appears to be a safe alternative. To determine if MB has potential detrimental effects on ovarian tissue, we compared the effect of MB and IC on human granulosa luteal cell (GC) function in vitro. Human oocyte-cumulus complexes were obtained during in vitro fertilization cycles and one to three were placed in an organ culture dish. After insemination with sperm, oocytes were removed the day after retrieval and the attached GC were washed daily for 3 more days by changing 2 mL of culture medium. All the dishes were treated with human chorionic gonadotropin (hCG) for the next 24 h and progesterone (P) production during this interval was taken as baseline. Test chemicals were added with hCG for the next 48 h with daily media changes. The P production during the last 24 h of chemical treatment was expressed as a percentage of the baseline. MB significantly reduced P production whereas IC did not appear to have any effect. Moreover, under inverted microscopy more than 90% of the GC cells contained several small bluish intracellular granules when exposed to 0.01% MB but not 0.01% IC. These results indicate that MB may be taken up and processed by GC cells and inhibits P production. This finding adds to previous reports on the use of in vitro GC assay to identify potential reproductive toxicants. The clinical significance of this preliminary study needs further investigation.