Supplementation with L-Carnitine Rescues Sperm Epigenetic Changes in Asthenospermic Male Semen with Altered Acetyl-L-Carnitine Levels

Objective:To investigate the relationship between the concentration of L-carnitine in semen and sperm parameters and investigate the epigenetic profile in sperm cell after L-carnitine usage.Methods:From February 2017 to February 2018,46 semen samples from asthenospermic males and 41 semen samples from healthy donors were acquired.Motility parameters were assessed using computer-assisted sperm analysis(CASA,n=78)and the DNA fragmentation index(DFI)was evaluated through flow cytometry(n=86),%DFI=%cells outside main population.Other oxidative stress markers,such as reactive oxygen species(ROS)levels(n=86)and the mitochondria DNA copy numbers,were detected(n=78).The concentration of L-carnitine and acetyl-L-carnitine was detected(n=82),and methylation was analyzed(n=30).After that,we collected 13 fresh semen samples from asthenospermic males and 23 fresh semen samples from healthy donors.These samples were used in a freeze-thaw model that was used to determine whether adding L-carnitine could change sperm progressive motility(n=23),apoptosis index(n=9),and methylation analysis(n=7).In total,we have done 13 asthenospermia samples for Western blot,and except for the poor Western result,we analyzed 6 samples for H3K9ac detection,7 samples for H3K9m3 and H3K27m3 detection,and immunofluorescence(n=3).Finally,we had recruited 30 volunteers,and they were given oral administration of L-carnitine for 3 months and then collected semen samples at different time points for methylation analysis.Results:The concentration of acetyl-L-carnitine is negatively correlated with the%DFI value(r^2=0.1090;P=0.0026),and the concentration of acetyl-L-carnitine is positively correlated with sperm forward motility(r^2=0.0543;P=0.0458)and ROS(r^2=0.1854;P<0.0001),and the acetyl-L-carnitine level is negatively correlated with%DFI in asthenospermia(r^2=0.1701;P=0.0066),and the level of acetyl-L-carnitine in asthenospermic semen is significantly lower than the normal group(P=0.0419).In addition,this study indicates that adding L-carnitine significantly improved sperm motility(P=0.0325)and reduced sperm apoptosis(P=0.0032).Importantly,Western blotting(P=0.0429)and immunofluorescence staining results showed that the addition of L-carnitine reduced H3K9Me3 methylation level in sperm,respectively.Furthermore,semen samples from asthenospermic patients had reduced methylation levels in a specific region(16^thP=0.0003;17^thP=0.0016)of the brain-derived neurotrophic factor(BDNF)promoter.The 16^th methylation decreased with age(r^2=0.1564;P=0.0306),and the 17^th methylation was decreased after treatment with L-carnitine for 28 days(P=0.0341).Conclusion:L-carnitine can reduce the%DFI and also affect the methylation of the histone modification marker in sperm as a possible epigenetic regulator.