The Effect Of Cryopreservation On DNA Intergrity, Apoptosis And Mitochondrial Membranf Potenntial Of Human Sperm

OBJECTIVES Sperm freezing technology has been widely used in reproduction, it can be used to provide human reproductive insurance, to supply qualified semen for infertile couples. However, the formation of ice crystals and oxidative stress during freezing would caused sperm damage (including the sperm membrane, DNA, movement rates, mitochondrial function, etc.), and DNA damage has resulted in a decline in sperm fertilization capacity. Mitochondria which is the sperm energy center is located in the sperm tail, and is closely related to sperm motility; At the same time, the mitochondrial apoptosis pathway plays a pivotal role in the process of apoptosis. The decline of mitochondrial membrane potential was considered to be the earliest apoptotic step. In this study, our main aim:1. The effect of cryopreservation on sperm motile ability, sperm morphology, membrane lipid peroxidation, DNA damage, mitochondrial membrane potential and apoptosis. 2. Compared with normal group, Caused cryopreservation differences in sperm?METHODS Donors who visited the Shandong province sperm bank during the period of March to October 2009 were recruited for the present study. The routine seminal parameters were evaluated according to the World Health Organization (WHO) Criteria (World Health Organization,1999). The variables taken into consideration were: volume, sperm concentration, total sperm number, motility, vitality and normal morphology. The ejaculates were used for normal sample only if the following requirements were fulfilled:less than 0.5*106 leukocytes/mL of semen; sperm concentration>20*106/ml,>30% of the spermatozoa with a normal morphological shape and>50% appearing progressively motile. Semen samples were collected by masturbation after 3-7 days of sexual abstinence. They were allowed to liquefy for 30 min at room temperature. The liquefied samples were then divided into two parts as follow:fresh semen and cryopreserved semen. We incorporated a set of functional sperm tests in every sample. These included tests of the mitochondrial membrane potential (MMP), sperm morphology,phosphatidylserine externalization (PS), DNA fragmentation and Oxidative Stress. The study was approved by the ethics committee for research involving human subjects.RESULTSâ‘ Normal group:The freezing-thawing process significantly influenced sperm morphology, MDA concentration, MMP, AN+/PI-and AN+/PI+.â‘¡Abnormal group:The results showed significantly differences in sperm morphology, DNA integrity, MDA concentration, MMP, AN-/PI-, AN+/PI-and AN+/PI+ between the fresh and frozen sample.â‘¢Compared with normal group, Cryopreservation caused significantly differences in sperm morphology, DNA integrity, AN+/PI-and AN+/PI+.â‘£Sperm morphology was significantly negatively correlated with the MDA concentration, the MDA concentration had significantly positive correlated with DNA integrity, The apoptotic spermatozoa'AN+/ PI-'positive correlated with sperm concentration, The percentage of'AN-/ PI-'spermatozoa positively correlated with sperm concentration,The level of'AN+/ PI+'spermatozoa negatively correlated with sperm morphology and MMP, MMP positively correlated with sperm motility.CONCLUSIONS Cryopreservation caused more damage in abnormal sperm than normal sperm. In working, if semen quality is low, the patients should be informed of the risk of cryopreservation.