Effects Of Different Freezing Methods On Human Sperm Fertilization Ability

Sperm cryopreservation can effectively preserve male fertility,and bring new hope for many infertile couples combined with assisted reproductive technology.After experts and scholars for many years unremitting research and exploration,there are many kinds of alternative freezing method,including slow programmable freezing,liquid nitrogen vapor freezing and sperm vitrification freezing,each method has both advantages and disadvantages.Slow programmable freezing effect is positive,but the instrument is expensive,the operation is time-consuming.Liquid nitrogen vapor freezing use the temperature gradient produced by liquid nitrogen vapor to freeze,it is widely used because of its simple operation and less time-consuming.However,the freezing rate of the method is not stable and it may cause damage to sperm.Vitrification freezing through the high cooling rate to reduce mechanical damage caused by intracellular ice,without the use of cryoprotectants,reduces the permeability damage to sperm,the sperm motility,viability and DNA integrity rate are improved after recovery.But the volume of frozen semen is small,it is usually used for oligospermatism and azoospermatism.Therefore,it is a hot spot to increase vitrification freezing volume for conventional semen.Objective: To compare the effect on sperm motility,vitality,morphology,acrosin activity and DNA integrity of post-thawed sperm after Liquid nitrogen vapor freezing and vitrification freezing with plastic cryogenic vial.And explore the effect of rapid semen freezing on sperm fertilization and the feasibility of the technique.Methods: Semen samples(n=35)were collected from patients seeking diagnostic assistance at reproductive medicine clinic in the first hospital of Hebei Medical University from September 2016 to January 2017.Patients were excluded with syphilis,AIDS,hepatitis B and other infectious diseases.Experiments were carried out on the patient consent,patients were 27~38 years old and abstinence time was controlled in 2~7 days.The semen samples were collected by masturbation and ejaculated into a sterile container,and allowed to liquefy and then immediately evaluated according to the WHO laboratory manual for the examination and processing of human semen(Fifth Edition).Inclusion criteria: liquefaction time ?1h,the semen volume ?1.5ml,the sperm concentration ?40×106/ml,activity rate ?40%.Fresh samples were processed using a density-gradient centrifugation method.Washed samples were divided into three groups: Group A,fresh semen;Group B,Liquid nitrogen vapor freezing with self-made yolk-glycerol-sodium citrate cryoprotectant;Group C,large volume rapid freezing with sucrose solution,the final concentration of sucrose was 0.25 M.In Group B and C,cryoprotectant should be added to semen slowly,side by side shaking,the proportion of semen and cryoprotectant was 1:1.After one week of storage,the semen samples were thawed,samples were removed from liquid nitrogen,put in water bath box at 36?for 10 minutes.Then the thawed samples were diluted in 1ml sperm rinse medium before being centrifuged at 200 g for 10 min.After discarding the supernatant,0.5ml of perm rinse medium were added in the precipitate and gently mixed.Sperm motility,vitality,morphology,acrosin activity,DNA integrity and the post-wash total mobile sperm count were detected respectively in group A,group B and group C.Results:1 The percentage of sperm motility,vitality,DNA integrity,acrosin content were lower than that of fresh semen after frozen,the deformity rate was higher than that of fresh group,the difference was statistically significant,P<0.05.2 In group C,the percentage of motile sperm,vitality and the post-wash total mobile sperm count were slightly higher than those of group B,but there was no significant difference,P>0.05.3 In group B,sperm malformation rate was higher than the group C,the difference was statistically significant,P<0.05.4 The sperm DNA integrity of group C was slightly higher than that of group B,but there was no statistical significance,P>0.05.5 In group C,crosin content was higher than that of group B,the difference was statistically significant,P<0.05.Conclusions: With the conventional plastic freezing tube as the carrier,and the sucrose solution as the cryoprotectant,large volume rapid vitrification freezing of semen after treatment could achieve the same effect as that of conventional freezing method.And the post-wash total mobile sperm count after recovery could meet the requirements of intrauterine insemination.Moreover,large volume rapid freezing is more convenient to operate,and does not need to prepare a special freezing carrier,at the same time,the operation time was less and the price was lower.