Human Sperm Vitrification Using Vertical Straw Freezing
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Solution Overview
Problem
Current sperm freezing methods, particularly non-aseptic ones, result in low post-devitrification motility recovery rates of 20-30%, while aseptic methods like WO 12028967 and CN 106962323 have inefficiencies in vitrification and devitrification times, and WO 12028967 lacks the use of permeable cryoprotectants.
Innovation Solution
An aseptic vitrification method using a vitrification medium with permeable and non-permeable cryoprotectants, combined with a vertical straw placement in liquid nitrogen, achieving rapid vitrification and devitrification times of 10 seconds and 50 seconds respectively, resulting in 80-95% motility recovery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-aseptic vitrification methods are used, then the procedure is simpler and faster, but the post-devitrification motility recovery is low (20-30%)
Solution Approach 1:
The invention changes the chemical parameters of the cryoprotectant solution by using a specific composition (10-30% permeable cryoprotectant, 5-20% non-permeable cryoprotectant, 1-5% permeable permeabilizer) to achieve both aseptic conditions and high motility recovery (80-95%) simultaneously
Solution Approach 2:
The invention introduces a permeable permeabilizer as an intermediary substance that facilitates the entry of permeable cryoprotectant into sperm cells while maintaining aseptic conditions, enabling both high motility recovery and sterile technique compliance
2Reliability
If WO 12028967 method is used with horizontal straw placement, then the procedure is aseptic, but the post-thaw motility is less than 5%
Solution Approach 1:
The invention inverts the straw placement orientation from horizontal (as in WO 12028967) to vertical position during liquid nitrogen immersion, which fundamentally changes the freezing dynamics and enables both aseptic conditions and high motility recovery (80-95%) to coexist
3Productivity
If slow freezing methods are used, then the procedure is simpler, but the vitrification time is long and motility recovery is low (20-30%)
Solution Approach 1:
The invention utilizes rapid phase transition of water to ice crystals during liquid nitrogen immersion, combined with the specific cryoprotectant composition, to achieve vitrification in approximately 10 seconds while maintaining high motility recovery (80-95%), replacing slow freezing with rapid freezing
Solution Approach 2:
The invention changes the temperature parameter by using liquid nitrogen at -196°C for rapid freezing, combined with optimized cryoprotectant concentrations (permeable: 10-30%, non-permeable: 5-20%), to achieve both fast vitrification (10 seconds) and high motility recovery (80-95%)
4Productivity
If CN 106962323 method is used with 4°C storage, then the procedure is simple, but the vitrification is incomplete and motility recovery is limited
Solution Approach 1:
The invention uses liquid nitrogen at -196°C to achieve complete vitrification through rapid phase transition, whereas CN 106962323 uses 4°C which only achieves partial freezing. This temperature difference enables complete vitrification and high motility recovery (80-95%) rather than limited recovery
Solution Approach 2:
The invention changes the temperature parameter from 4°C (CN 106962323) to -196°C (liquid nitrogen), and combines it with optimized cryoprotectant composition, to achieve complete vitrification and high motility recovery (80-95%)
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly enhances sperm motility recovery post-devitrification to 80-95%, improving efficiency and reducing time compared to existing methods.
Implementation Method 1
The invention provides a simple, fast and effective sperm vitrification method
Implementation Method 2
immerse the sealed straws containing the sperm in liquid nitrogen
Implementation Method 3
a permeable cryoprotectant and a non-permeable cryoprotectant
Implementation Method 4
devitrify the sample in a devitrification medium at 37° C.
Implementation Method 5
devitrification time about 50 seconds
Data Source
AI summary
A method for human sperm vitrification is disclosed, which includes providing liquefied human sperm; having a vitrification medium, where the vitrification medium has (a) a sperm buffer, and (b) a cryoprotectant mixture including a permeable cryoprotectant and a non-permeable cryoprotectant; mixing the liquefied sperm with the vitrification medium; providing a sperm straw and loading the straw with 0.25 to 0.5 mL of the obtained mixture; sealing the straw with heat; and placing the straw vertically in a container with liquid nitrogen to vitrify the mixture. A portable kit is also disclosed, which makes it easy to implement the method. The kit includes a vitrification medium, some straws, a container, usage instructions, among others.