The Study Of Human Sperm Motility Bioassay In IVF Laboratory Quality Control And Freeze Methods

The assisted reproductive technologies, known as ART consist of therapies for infertility such as in vitro fertilization and embryo transfer (IVF-ET), intracytoplastic sperm injection (ICSI), preimplantation genetic diagnosis (PGD) have been widely accepted throughout the world and are using technologies for treating infertility. The major concern of an IVF is to obtain high pregnant rate. This can be done by producting high quality embryo. One of the factors that affect embryo quality is the in vitro milieu. It is not surprise that even low levels of toxicants present during embryo culture will dimish the viability the embryos. Many components used for IVF can impair the growth of human embryos. To monitor the complex in vitro system, quality control methods are used to detect toxin in related items that might directly or indirectly contact embryos is required to maintain the desired pregnancy rate. It is important to realize that toxic substances can be transferred during brief contact with any equepment. The optimal culture of human embryos in an IVF program demands the selection of non embryo toxic equipment and reagents objective, quality control strategies have been developed that assess the performance of specific areas with the laboratory. The toxicity of different reagents and materials used in an IVF program has been evaluated by using a range of bioassay that include the one-cell and two cell mouse embryo assay (MEA), human sperm survival assay (HSSA), hamster sperm motility assay (HSMA), human amniotic fluid cell culture(HAFC) and hybridoma cell culture (HICC).Successful cryopreservation and thawing of human semen has been done for decades in assisted reproduction field. The cryopreservation semen has been the best way to keep human sperm in long period. However, the motility and concentration of frozen sperm is reduced by as much as 50% after freezing and thawing. It is generally believed that multiple factors may contribute to the cryodamage of sperm cells, causing a decline in motility. Attention has been given to defining mechanisms%of cellular damage, which may include ice crystal formation and osmotic force. Also whether the cryoprotectant is safe to sperm in process of freezing asked by some embryologists.The purpose of these projects was to evaluate the sensitivity and value of human sperm motility assay as a quality control method in IVF laboratory; Detect embryo toxin of some items used in IVF laboratory; Search an the optimal freeze method.AbstractObjective:1. To determine the sensitivity of human sperm survival assay to using known concentrations of potential toxin of formalin; Whether or not the lowest level toxin can be detected by this assay.2. To test the quality of three types culture tubes used commonly in IVF program; To understand the effects of albumin bovine serum and light mineral oil on sperm quality during culture;3. To identify the optimal procedures for freezing low numbers of sperm in cryoprotectant Test-Yolk-Buffer (TYB). To determine which freezing method was better to sperm motility for freezing direct plunging into liquid nitrogen tank or 20 min exposure to liquid nitrogen vapor first.Methods:The fresh semen was obtained from health males at andrology laboratory by masturbation. Sperm were processed on a gradient column of Isolate medium and PBS medium.In experiment 1 The medium with two concentrations of formalin and control medium were added to the Falcon culture tube and culture dish with 6 wells contained medium with or without bovine albumin serum and with or without light mineral oil. The endpoint was the index of motility at 24hrs and 48hrs divided by the motility prior to exposure to the tubes.In experiment 2 In 3types of culture tubes contained medium with or without light mineral oil and with or without bovine albumin serum, the sperm were exposed to the each culture tubes. The endpoint was the index of motility at 24hrs and 48hrs divided by the motility prior to exposure to the tubes oil or protein.In experiment 3 After semen were mixed with TYB cryoprotectant medium at 5min,15min, 30min and 60 min, checked the sperm motility. Two methods were used to freeze the cry vials: immediate plunging into liquid nitrogen or 20 min of exposure to liquid nitrogen vapor followed with direct plunging.The sperm motility was counted under the microscope.Statistical analysis: Date from the 3 experiments was subjected to factorial design, using analysis of variance (ANOVA) and was performed with the statistical package for social sciences (SPSS) 12.0.Results:1. The everage sperm motility index was decreased in HTF medium with 0.25% formalin at 24hrs, the higher formalin concentration was, the lower sperm motility had; the longer time contact with formalin, the less sperm counting was (PO.001);2. The average sperm motility index of 7ml tissue culture tube made in Demark was 0.677±0.335, higher than another two types culture tubes made in U.S.A: 0.551±0.317, 0.595±0.327 (PO.001); no difference was found between two culture tubes of American; When the sperm cultured in the medium with albumin bovine serum or light mineral oil, the average sperm motility index was higher than without albumin bovine serum or light mineral oil (P<0.001); The average sperm motility index in cultured 48hrs was lower than that of 24hrs;3. After mixed up with TYB cryoprotectant, the average sperm motility index was lower in 4 time stages, meanwhile, the motility of sperm in 15min and 60min was significantly decreased (P<0.001) and 30min was alittle recoved;There was a significant difference on freezing methods, the vapor first was better than directly plunge into liquid nitrogen (P<0.001).Conclusion:1. The sperm motility bioassay is a sensitivity quality control method to detect the components used in IVF laboratory; the sperm motility significantly decreased when sperm were cultured in medium containing 0.25% formalin at 24hrs.2. 7ml tissue culture tube made in Demark can be used to culture human embryo,other two type tubes can be used in retriving eggs; the best culture environment was that the sperm was cultured in medium with albumin bovine serum or light mineral oil at 24hrs.3. Semen can not be placed in TYB medium too long time in the room temperature 30min was a time point to distinguish the sperm quality; Sperm frozen in vapor liquid nitrogen first, then place into liquid nitrogen can get better motility recovered after thawing.