摘要:
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 (
r2 = 0.1090;
P = 0.0026), and the concentration of acetyl-L-carnitine is positively correlated with sperm forward motility (
r2 = 0.0543;
P = 0.0458) and ROS (
r2 = 0.1854;
P < 0.0001), and the acetyl-L-carnitine level is negatively correlated with %DFI in asthenospermia (
r2 = 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 (
r2 = 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.