Effects Of L-carnitine On DNA Fragmentation,Mitochondrial Membrane Potential And Acrosome Reaction During Human Sperm Cryopreservation

BackgroundHuman sperm cryopreservation is one of the important techniques in the field of assisted reproduction.The quality of frozen spermatozoa in the sperm bank is the key to the succeed of assisted reproductive technology.In addition,it is common that it is difficult for men to obtain sperm by themselves on the day of oocyte retrieval due to mental stress in clinical work.Worse still,epididymal puncture is required to obtain sperm.If such patients can pre-freeze semen,they can avoid the injury caused by puncture.Therefore,how to improve the effect of sperm cryopreservation is a hot topic in the field of assisted reproduction.In cryopreservation,reactive oxygen species will be produced,which does harm to sperm cell membrane.Sperm freezing leads to the formation of intracellular ice crystals and sperm motility decrease,which has also caused sperm DNA and mitochondrial dysfunction and a series of damage.Many scholars are looking for different protective agents to minimize this damage.Currently,L-carnitine has been applied to the field of reproductive medicine pretty widely.It can effectively promote the occurrence of sperm and improve the sperm's ability to exercise and fertilize.In addition,sperm motility can be improved by L-carnitine.Researchers abroad have added L-carnitine to frozen spermatozoa.The results show that adding L-carnitine can effectively improve sperm motility and reduce DNA fragmentation,and at the same time lead to decrease in the rate of apoptosis and division of sperm cells.No similar research has been found in China.Thus,this topic intends to further explore the protective effect of L-carnitine on human sperm during cryopreservation.ObjectiveTo explore the effects of different concentrations of L-carnitine to conventional cryoprotectants on sperm PR,motility,DNA fragmentation index,mitochondrial membrane potential and acrosome reaction during human sperm cryopreservation.Whether L-carnitine can improve the effect of sperm cryopreservation and choose the best concentration of L-carnitine.Materials and MethodsForty patients were randomly selected from the June 2017 to November 2017 at the Reproductive Center of the Second Affiliated Hospital of Zhengzhou University.Experiments were carried out on the patient consent.Abstinence time was controlled in 2⁷ days of all patients.In addition,the semen samples were collected by masturbation method,and placed in a 37?water bath liquefaction.After liquefaction,the sperm samples were routinely analyzed according to the fifth edition of the WHO laboratory manual for the examination and processing of human semen.Inclusion criteria:liquefaction time?1h,the semen volume?3ml,the sperm concentration?50×10⁶/ml,motility?50%.With the conventional 1.8ml plastic freezing tube as the carrier,the sperm homemade glycerol-yolk-sodium citrate cryoprotectant were commonly used by human sperm bank.All semen samples were fully mixed and divided into five groups:group A:fresh semen as control;group B0:conventional cryoprotectant;group B5:cryoprotectant with 5mmol/L L-carnitine;group B10:cryoprotectant with 10mmol/L L-carnitine;group B20:cryoprotectant with 20mmol/L L-carnitine.The ratio of semen and cryoprotectant was 3:1 and the semen samples were frozen by liquid nitrogen vapor.Spermatozoa thawing after 48 hours.Computer-aided sperm analysis was used to detect the PR and viability of spermatozoa before and after cryopreservation.Using flow cytometry detects DNA fragmentation,mitochondrial membrane potential and acrosome reaction in each group.Results1.The PR,motility and mitochondrial membrane potential after freezing were significantly lower than those before freezing.The sperm DNA fragmentation index was higher than that before freezing.In addition,the difference was statistically significant?P<0.05?.2.There was no difference in the likelihoods of the PR,motility between four frozen groups.?P>0.05?;3.The sperm DNA fragmentation index of spermatozoa were as follows:cryoprotectant added with 10 mmol/L L-carnitine group<cryoprotectant added with20 mmol/L L-carnitine group<cryoprotectant added with 5 mmol/L L-carnitine group<conventional cryoprotectant(B₁₀<B₂₀<B₅<B₀).The B₁₀ and B₂₀ groups were significantly lower than the B0 and B5 groups.The difference was statistically significant?P<0.05?;4.The mitochondrial membrane potentials of spermatozoa were as follows:conventional cryoprotectant<cryoprotectant added with 5 mmol/L L-carnitine group<cryoprotectant added with 10 mmol/L L-carnitine group<cryoprotectant added with20 mmol/L L-carnitine group(B₀<B₅<B₁₀<B₂₀).The B₁₀ and B₂₀ groups were significantly higher than the B0 and B5 groups.The difference was statistically significant?P<0.05?;5.There was no significant difference in acrosome reaction between four frozen groups?P>0.05?.Conclusions1.Sperm cryopreservation would lead to frozen injury,which reduced PR,sperm motility,mitochondrial membrane potential.Besides,sperm cryopreservation also lead to sperm DNA fragmentation index increase;2.Cryoprotectant added with 10 mmol/L L-carnitine or 20 mmol/L L-carnitine group will reduce the sperm DNA fragmentation index and mitochondrial membrane potential decreased.In this way,the effect of sperm cryopreservation can be improved.10mmol/L of L-carnitine can be used as an active ingredient to protect spermatozoa in human sperm cryopreservation.