In vitro effect of levonorgestrel on human sperm function

M.J. MUNUCE; J. NASCIMENTO; G. ROSANO; A. FAUNDES; L. BAHAMONDES

Praga, República Checa

Otro; 22nd Annual Meeting of the European Society of Human Reproduction and Embryology; 2006

European Society of Human Reproduction and Embryology

Introduction: The levonorgestrel-releasing intrauterine system (LNG-IUS) exerts its contraceptive effect by interfering with sperm transport, ovulation and fertilization However, a direct effect of the steroid on sperm function cannot be discarded. The aim of this study was to evaluate whether LNG, at doses comparable to that measured in the uterus during the use of the LNG-IUS, affects the detection of D-mannose-binding sites or zona pellucida (ZP) receptors on human spermatozoa. The association with acrosomal status was also investigated. Materials and methods: Semen samples from normozoospermic donors were used for the study (n=17). Seminal plasma was removed by layering the semen on a Percoll gradient (90–50%). Motile spermatozoa were incubated under capacitating conditions for 22 h at 37 ºC under 5% CO2 in air. A stock solution of LNG was prepared by dissolving the hormone in ethanol (mg/ml). Capacitated spermatozoa were exposed to 1 or 10 ng/ml of LNG or control medium for 30 min. To exclude any toxic effect on sperm function, sperm viability was evaluated by eosin Y at the end of all procedures. D-mannose-binding sites were detected on sperm surface using D-mannosylated-BSA conjugated with fluorescein isothiocyanate (Man-FITC-BSA). Each spermatozoon was categorized according to the pattern of the fluorescent signal as follows: pattern I (tail and midpiece, unspecific), pattern II (whole acrosomal cap plus midpiece, specific) or III (equatorial/post equatorial plus midpiece, specific). Acrosomal status was analyzed using the Pisum sativum technique. Double labeling with Man-FITC-BSA and rhodamine-conjugated Pisum sativum agglutinin was also performed. Data were expressed as mean±SEM and were analyzed using the one-way ANOVA and the Tukey–Kramer test or the c2 test. A p-value<0.05 was considered as significant. Results: LNG did not affect the percentage of viable cells at the doses assayed (>74% viable cells). When capacitated spermatozoa were exposed to LNG a significant increase in the percentage of spermatozoa showing pattern III was observed with respect to controls (control: 25±5% vs. LNG; 1 ng/ml: 34±7%* vs. LNG; 10 ng/ml: 36±5%*, n=5, *p<0.01 with respect to control). The proportion of sperm showing pattern II was not modified by LNG, since values in treated spermatozoa remained similar to controls (<10%). When the ability to stimulate the acrosome reaction was investigated, results showed that in the presence of LNG there was a significant increase in the proportion of reacted cells with respect to controls (control: 5.0±0.3% vs. LNG; 1 ng/ml: 6.3±0.4%* vs. LNG; 10 ng/ml: 7.8±0.4%*, n=12 *p<0.01 with respect to control). Double staining showed that although all cells presenting pattern II of D-mannose receptor had intact acrosome, those showing pattern III were reacted (163 cells). Conclusion: It is assumed that zona binding and the occurrence of the acrosome reaction are both prerequisites for fertilization which usually occurs on the ZP. Once reacted, human spermatozoa do not further bind to the ZP. In this study we observed evidence that in vitro exposure of capacitated spermatozoa to the assayed doses of LNG increased the proportion of spermatozoa with fewer chances of interacting with the ZP. Further studies should be carried out to confirm whether this mechanism is part of the contraceptive action of the LNG-IUS.