In vitro effect of levonorgestrel on sperm fertilizing capacity and mouse embryo development

Abstract

The objectives of this study were to assess the expression of α- d-mannose binding sites in human spermatozoa, human sperm–oocyte interaction and the development of early stages of mouse embryo in the presence of levonorgestrel (LNG). Semen samples were obtained from 16 normozoospermic men. Spermatozoa were separated by Percoll gradient and incubated overnight for capacitation. The kinetic analysis of the expression of α- d-mannose binding sites was determined at 0, 4 and 22 h and in 22 h-capacitated spermatozoa that had been exposed to 1, 10 or 100 ng/mL of LNG or to a control medium for 30 min. Sperm binding sites for α- d-mannose were detected using commercial α- d-mannosylated bovine serum albumin conjugated with fluorescein isothiocyanate. To evaluate sperm–oocyte interaction, each oocyte was placed in a 100-μL droplet containing one of the three doses of LNG or control medium and inseminated with 1.0×10 5 motile spermatozoa/mL, after which the number of bound spermatozoa was evaluated. A total of 157 two-cell embryos recovered from eight mice was pooled and assigned randomly to treatment (1, 10 or 100 ng/mL of LNG) or control groups. There was a significant increase in the expression of specific α- d-mannose binding sites (Patterns II and III) during the incubation of spermatozoa under capacitating conditions. In the presence of LNG, results showed that there was no significant difference in the expression of specific α- d-mannose binding sites (Patterns II and III) at any LNG concentration tested compared with those spermatozoa in control medium. None of the LNG concentrations were capable of modifying the number of spermatozoa tightly bound to the human zona pellucida. There was no association between the presence or absence of LNG or the different doses of LNG evaluated and mouse embryo development. In conclusion, the hypothesis that in vitro exposure to LNG could interfere with sperm function and could contribute to the mechanism of action of this form of contraception was not confirmed but cannot be ruled out by the results of this study.