Explore The Spermicidal Effect Of Ozonated Olive Oil As A Contraceptive Agent And Its Mechanism

Part I Evaluate the spermicidal effect of ozonated olive oil in human sperm in vitroObjective: To evaluate the spermicidal activity of ozonated olive oil by observing the effect of different concentrations of ozonated olive oil on human sperm motility.Methods: Normal semen samples were selected according to WHO laboratory manual for the examination and processing of human semen standards. After optimized by a discontinuous density-gradient centrifugation, sperm were co-incubated with ozonated olive oil of different concentration(3,4,5,6,7 μg/m L) at the ratio of 1:1(v/v) for 20 s. The group that completely lost motility within 20 s was tested for motility revival experiments subsequently. At time 0, 5, 10 and 30 min intervals up to 60 min of incubation the solution was observed if there was any recovery of motility.Results: The sperm motility were(30.11 ± 4.15)%,(20.11 ± 5.18)%,(7.50 ± 2.96)%,(1.70 ± 1.70)%, 0, respectively, after treated with ozonated olive oil at different concentration(3,4,5,6,7 μg/m L) for 20 s. Each group was much lower when compared with olive oil [(72.70±2.42)%] and Ham’s F-10 group[(77.90±2.10)%](both P<0.01). There was no significant difference between olive oil and Ham’s F-10 group( P>0.05). The MEC of ozonated olive oil required to 100% immobilize sperm in 20 s was(5.80±0.25) μg/m L. None of the human sperm, once immobilized, regained motility after removing the ozonated olive oil and resuspending in Ham’s F-10 for 60 min.Conclusion: Ozonated olive oil dose-dependent inhibited sperm motility. The MEC for 100% immobilize sperm in 20 s was found to be 6 μg/m L. Part II The spermicidal mechanism research of ozonated olive oil on human spermObjective: To explore the spermicidal mechanism of ozonated olive oil by detect its effect on human sperm viability, ultra-structure, function and DNA integrity.Methods: Normal semen samples were selected and optimized by a discontinuous density-gradient centrifugation, sperm were co-incubated with Ham’s F-10, olive oil and ozonated olive oil(6 μg/m L) at ratio of 1:1(v/v) for 20 s. Washed with Ham’s F-10 and sperm pellet was collected.(1)Sperm viability was evaluated by Eosin-Nigrosine staining.(2) Uranyl acetate and lead citrate double staining was used to observe the change of sperm ultra-structure by transmission electron microscopy.(3)sperm mitochondrial status was evaluated by testing MMP.(4) Calcium ionophore A23187-induced acrosome reaction(AR) was assessed by and PSA-FITC and Hoechst 33258 double florescent staining.(5) Sperm chromatin structure assay was used to evaluate sperm DNA integrity.Results:(1) Sperm viability: The value of sperm viability after treatment in ozonated olive oil group [(31.09±3.30)%] was significantly lower than that in olive oil [(87.64±1.01)%] and Ham’s F-10 groups [(89.73±0.84)%](both P<0.01).(2) Sperm ultra-structure: In Ham’s F-10 group sperm displayed intact plasma and mitochondria arranging in order, but in ozonated olive oil group it showed discontinuous and swelling plasma membrane and disordered mitochondria.(3) MMP: The value of normal MMP in ozonated olive oil, olive oil and Ham’s F-10 group was(35.63±7.64)%,(77.5±2.90)% and(86.17±1.25)%, respectively. The MMP in ozonated olive oil was significantly lower than that in Ham’s F-10 and olive oil groups(both P<0.01).(4) AR: The result of A23187-induced AR in ozonated olive oil, olive oil and Ham’s F-10 group was(19.01±2.92)%,(41.01±5.24)% and(43.83±4.75)%, respectively. The result in ozonated olive oil was lower than that in Ham’s F-10 group(P<0.01).The spontaneous AR in the three groups were(12.56±1.10)%,(12.84±1.43)% and(11.73±1.92)%, respectively, no significant difference of spontaneous AR was found among these three groups( P>0.05).(5) DNA integrity: The value of DFI was(14.70±2.61)%,(11.01±2.07)% and(7.52±0.77)%, respectively. The result in ozonated olive oil group was higher than that in Ham’s F-10 group(P<0.01).Conclusion: The findings demonstrated that ozonated olive oil dramatically disrupted human sperm membrane structure, sperm function and sperm DNA integrity. These indicated a potential spermicidal effect and may be acted as a promising vaginal contraceptive method.