Analysis Of Factors Affecting Sperm Cryopreservation And Preliminary Study Of The Mechanisms

Section 1. Analysis of factors affecting sperm cryopreservationAims:In order to provide the basis for improving the recovery rate of human frozen spermatozoa. We would analyze factors affecting sperm cryopreservation including ejaculate collection season, abstinence time, sperm quality, ROS level in seminal plasma, sperm DNA injury and seminal plasma from other donors.Materials and methods:1. A total of 14190 semen samples donated at human sperm bank of Zhejiang Province (Esastern China) between September 2006 and June 2011 were collected from 1624 donors. Semen was evaluated according to WHO standard procedures and sperm concentration, progressive motility, sperm morphology, ejaculate collection season, and abstinence time were recorded. After freezing and thawing, the progressive motility was assessed.2. A total of 34 semen samples were collected from 28 semen donors. Each semen sample was divided into two parts. One was added hypoxanthine (HX) and xanthine oxidase (XO) to generate reactive oxygen species (ROS), the other added Phosphate buffer saline (PBS) as the control group. Semen samples were frozen and thawed after being incubated for 1 hour, and the recovery rate was compared between the treatment group and control group.3. A total of 55 semen samples were collected and the DNA fragmentation index (DFI) was detected. Semen samples were divided into two groups according to DFI (＜15% and≥15%). The recovery rate was compared between the two groups after freezing and thawing.4. A total of 25 semen samples were collected. Each semen sample was divided into two parts. One was added the seminal plasma from donor's semen with high recovery rate as the treatment group, the other was added their own seminal plasma as the control group. The recovery rate was compared between the two groups after freezing and thawing. Results:1. (1) The progressive motility recovery rate was the lowest in semen donated in summer (67.1%), with statistically significant differences from spring (March—May) (69.2%), autumn (September-November) (69.0%) and winter (December-February) (69.2%)(P＜0.05). There was no significant difference between spring, autumn and winter (P＞0.05). (2) The progressive motility recovery rate decreased significantly with the prolonged abstinence time. It was lowest in the group with abstinence time for 7 d (P＜0.05) and was highest in the group with abstinence time for 3 d (P＜0.05). (3) The progressive motility recovery rate of frozen spermatozoa rose significantly with increasing sperm concentration. Compared with other groups, the progressive motility recovery rate was lowest in the group for sperm concentration≤60×106ml-1 (P＜0.05) and was highest in the group for sperm concentration>120×106ml-1(P＜0.05). Correlation analysis also showed that the progressive motility recovery rate significantly correlated with sperm concentration. (4) The progressive motility recovery rate rose significantly with the improvement of progressive motility of semen samples. It was lower in the group with progressive motility≤50%than that in other two groups with progressive motility for 51%～60%and＞60%(P＜0.05). (5) The progressive motility recovery rate rose significantly with better sperm morphology. It was lowest in the group for normal morphology≤10%(P＜0.05) and was highest in the group for normal morphology＞25%(P＜0.05). Correlation analysis also showed that the progressive motility recovery rate significantly correlated with the percentage of normal morphology spermatozoa.2. The frozen-thawed progressive motility and the progressive motility recovery rate (11.44%±9.11%,32.77%±18.85%) in the treatment group with HX+XO were lower than those in the control group (24.03%±9.0%,47.62%±15.25%),P＜0.05.3. The frozen-thawed progressive motility and the progressive motility recovery rate (20.63%±1.75%,43.94%±16.63%) in the group for DFI＜15%were higher than those in the group for DFI≥15%(12.92%±8.58%,29.80%±15.39%), P＜0.05. Correlation analysis also showed that the progressive motility recovery rate negatively correlated with DFI significantly.4. The frozen-thawed progressive motility and the progressive motility recovery rate (28.80%±14.52%＞58.62%±34.12%) in the treatment group added the seminal plasma with the high recovery rate were higher than those in the control group added their own seminal plasma (21.44%±15.57%,43.77%±38.92%),P＜0.05.Conclusions:The progressive motility recovery rate would be affected by ejaculate collection season, abstinence time, sperm concentration, progressive motility, sperm morphology, ROS level in seminal plasma, sperm DFI and seminal plasma. Our results indicated that some above factors could be valuable in predicting the progressive motility recovery rate of human frozen spermatozoa. The progressive motility recovery rate would be greatly improved by adjusting these factors. Therefore, the number of qualified donors and the amount of frozen semen would rise. Section 2. The role of proteins in sperm and seminal plasma on sperm cryopreservationAim:In order to detect differentially expressed proteins affecting human sperm freezing tolerance, investigate the damage mechanisms of sperm freezing at the proteomic level and provide the basis for improving technology of sperm freezing, we performed a comparative study of human sperm and seminal plasma proteins with high and low recovery rate of frozen spermatozoa using the two-dimensional electrophoresis and mass spectrometry technology.Materials and methods:Five semen samples with high recovery rate of frozen spermatozoa (83.6%±4.8%) and five with low recovery rate of frozen spermatozoa (26.1%±7.3%) were obtained from sperm donors. Spermatozoa and seiminal plasma were collected by up-swimming and high-speed centrifugation, respectively. Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) were used to investigate the differences in the proteome of human sperm and seminal plasma between high and low recovery rate of frozen spermatozoa.Results:A total of 61 differentially expressed proteins spots were found in the sperm (n=26) and seminal plasma (n=35) with high and low recovery rate of frozen spermatozoa.23 of them were chosen to be analysed by mass spectrometry technology. The results showed that these proteins may have important roles in sperm maturation, sperm movement, energy metabolism, cell cytoskeleton, sperm DNA repair and other activities.Conclusions:Proteins in sperm and seminal plasma associated with sperm maturation, sperm movement, energy metabolism, cell cytoskeleton and sperm DNA repair would affect human sperm freezing tolerance...