Comparison Of Cryopreserved Human Sperm In Vapor And Liquid Phases Of Liquid Nitrogen

Objectives:Cryopreservation of sperm is one of the fundamental techniques in reproduction, it can be used to provide human reproductive insurance, to supply qualified semen for infertile couples. The technical aspects of sperm cryopreservation have significantly improved over the last few decades. However, the formation of ice crystals and oxidative stress during freezing would caused sperm damage (such as the sperm membrane, DNA, movement rates, mitochondrial function), and DNA damage has resulted in a decline in sperm fertilization capacity. To preserve the viability of sperm, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the nonsterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. Cryopreservation of sperm is an extremely important issue in the field of male infertility as freezing can have detrimental effects on a variety of sperm functions, some of them not accessible to the traditional semen quality analysis. Considering the significant potential for cross-contamination during semen cryostorage using current methodology, and consequently the finite (but probably unquantifiable) risk of cross-infection during ART procedures, our study was to compare the effects of cryopreserved sperm in vapor and liquid phases of liquid nitrogen on sperm motility, morphology, sperm function and post-thaw DNA integrity, to investigate the effects of cryopreservation time on sperm function, and to establish a standard protocol to optimize sperm motility recovery.Methods:Donors who visited the Shandong provincial human sperm bank during the period of March to October 2009 were recruited for the present study. The routine seminal parameters were evaluated according to theWorld Health Organization (WHO) Criteria (World Health Organization,1999). The variables taken into consideration were:volume, sperm concentration, total sperm number, motility and normal morphology. The ejaculates were used for normal sample only if the following requirements were fulfilled:sperm concentration>60×106/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. Forty-eight semen samples with normal motility and sperm count were collected from 48 men who donated sperm for research. They were allowed to liquefy for 30 min at 37℃CO2 incubator. Each semen sample was divided into two aliquots. Samples were frozen with static-phase vapor cooling. One aliquot was plunged into liquid nitrogen (-196℃) and the other was stored in vapor-phase nitrogen (-167℃) for 2 days,7 days,30 days and 180 days. Thawing was performed at 37℃water bath incubator. The fresh and thawed aliquots were also assessed by artificial counting method of motility and fluorescence microscopy (after Acridine Orange staining, AO). The sperm morphology was determined by Diff-Quik staining procedure. Statistical Analysis Results were analyzed by using the Statistical Package for the Social Sciences software (version 13; SPSS, Chicago, IL). The data are presented as mean±SD. Data with a normal distribution were analyzed by paired Student's t-test. Data without a normal distribution, such as postthaw sperm with normal morphology, were analyzed by using the Wilcoxon signed-ranks test. P<0.05 was considered to indicate statistically significant differences. One protocol (which includes your data monitoring plan) and consent forms are approved by the IRB of our hospital.Results:Sperm quality was found to deteriorate after cryopreservation. Most of the parameters of postthaw sperm stored in the vapor phase(such as motility, normal morphology and DNA integrity) were not significantly different from those stored in the liquid phase of liquid nitrogen. Most of the parameters of postthaw sperm stored in the vapor phase tend to decrease with time, the results do not quite achieve statistical significance.Conclusions:Cryopreservation of human sperm in a vapor phase of liquid nitrogen was comparable to cryopreservation in a liquid phase of liquid nitrogen. The storage of human semen in a vapor phase of liquid nitrogen, without direct contact with liquid phase, may represent a useful alternative for the effective storage of human semen. All of their procedures at the outset must be critically examined including cryopreservation time and operation frequency in order to eliminate the risk of cross-contamination and improve the quality of sperm.