Elevated Seminal (SP) Plasma Lead (Pb2+) Levels Result in Significant Impairment of Human Sperm Function and Reduced Artificial Insemination (AI) Cycle Fecundity Rates ( f)

Abstract

Objectives: Under an IRB approved protocol, 2-3 blood plasma (BP) and 2-9 SP specimens were collected over a six month period from AI semen donors (n=14) and from an unselected population (n=13) answering an advertisement for research participation. The Pb2+ levels in these specimens had low (<9.5%) intra-male variation but varied between men over a wide range (e.g., <10 μg/L to ∼1200 μg/L). Pb2+ in BP and SP were positively correlated (Spearman correlation [SC], r=0.9, p<0.0001). Comparable BP/SP Pb2+ levels were previously seen in IVF patients, whose increased Pb2+ levels: [1] were negatively correlated with conventional insemination fertilization rates (SC, n=89, r=−0.6, P<0.0001), and [2] were unrelated to serum FSH, LH or testosterone levels. Preliminary findings also suggested that Pb2+ levels and f were negatively related (n=3, SP Pb2+ <250 μg/L, f >16% vs. n=2, SP Pb2+ >700 μg/L, f <6%; t-test, P<0.004). Therefore, the purpose of this study was to determine if increased Pb2+ levels were related to levels of other toxic metals, to decreased expression of sperm biomarkers for high IVF fertilization rates, and to f. Design: A double-blind study of cadmium (Cd2+) levels, sperm function and f (in 341 AI attempts) in specimens previously examined for Pb2+ content. Metal ion effects were modeled in vitro by exposure of fertile donor sperm (n=24) under capacitating conditions to increasing doses of exogenous Pb2+ (0–5180 μg/L). Materials and Methods: Cd2+ in BP and SP was assayed by graphite furnace atomic absorption spectroscopy. WHO (1992) semen parameters were recorded for fresh semen. Motile sperm populations were obtained from frozen/thawed semen by Percoll density gradient centrifugation and were examined for surface expression of mannose (Man) receptors (MR) and non-nuclear progesterone (P) receptors (NNPR), spontaneous and agonist (free Man and P)-stimulated acrosome reactions (AR). Results were compared using SC and repeated measures analysis of variance (RMANOVA) models. Results: BP and SP Cd2+ levels were more tightly clustered than Pb2+ (range, 0.081 μg/L to 0.161 μg/L). Pb2+ and Cd2+ in BP and SP were positively correlated (SC, respectively, n=11, r=0.7, P<0.01 and n=13, r=0.7, P<0.007). SP Pb2+ levels and sperm count, motility, percentage normal morphology and NNPR were not related. In contrast, significant inverse relationships existed between SP Pb2+ levels and MR (SC, n=12, r=−0.5, P<0.007), Man AR (SC, n=15, r=−0.5, P<0.04) and P AR (SC, n=26, r=−0.4, P<0.03). SP Pb2+ and spontaneous AR were positively correlated (SC, n=16, r=0.6, P<0.01). SP Pb2+ levels and f showed a strong inverse relationship (SC, n=24, r=−0.6, P<0.02). In vitro exposure of fertile donor sperm to exogenous Pb2+ resulted in a dose-dependent decrease in MR (RMANOVA, P<0.01), Man AR (RMANOVA, P<0.0008) and a dose-dependent increase in spontaneous AR (RMANOVA, P<0.0007). Conclusions: SP Pb2+ in subjects studied are representative of the general population. Within the AI group, selected for normal WHO semen parameters, donors with high SP Pb2+ had significantly lower fertility. The correlation between Cd2+ and Pb2+ suggests other metal toxicants may play a role. But in vitro modeling shows Pb2+ alone is sufficient to induce the same characteristics (increased spontaneous AR, decreased MR, decreased agonist-induced AR) in fertile sperm as seen in vivo in subjects with high SP Pb2+. We suggest that potential donors be screened for Pb2+ levels and sperm biomarker expression prior to acceptance into an AI program. These methods may also prove useful in directing ART patients to conventional IVF or to ICSI. (Support: NIH Grant No. ES 06100 to S.B.)