U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

[Narrator] The research focuses on a method for sperm optimization, aiming to determine whether UHS technique offers advantages in preparing high quality sperm with a low DNA Fragmentation Index. Microfluidic technology has been utilized to optimize the selection of high quality sperm with lower DFI. The main limitation of the UHS method is its high reliance on the self-swimming ability of high quality sperm. UHS method is not suitable for patients with completely immobile sperm. Thigmotaxis plays a crucial role in sperm separation. The U-shaped orbit has a longer boundary, which facilitates the free movement of high quality sperm, enabling their complete separation from immobile sperm, impurities, and other components. Compared to the DGC technique, the UHS lane method involves fewer manual operation steps for laboratory personnel, and high quality sperm obtained through the UHS lane method do not undergo repeated centrifugation procedures, resulting in a lower DFI. To begin, add 20 microliters of fertilization medium to the operating dish to create U-shaped tracks. Suck 10 microliters of fertilization medium with a pipette to create a circular droplet at the left starting point of the U-shaped track. Using fertilization medium, create two buffer droplets of 10 microliters each at intervals along the left middle section of the U-shaped track. Connect all droplets with fertilization medium to form a continuous track. Use 2.5 microliters of polyvinyl pyrrolidone, or PVP, to create a long strip of liquid droplet on the right side of the U-shaped track. Make six microdroplets using the oocyte processing medium on the right side of the PVP strip. Add seven milliliters of culture oil to the dish, ensuring the oil completely covers the highest point of the droplets. Place the dish in a 37 degrees Celsius, 6% carbon dioxide incubator overnight to equilibrate. After collecting the semen, measure its volume using the weighing method, ensuring accuracy to 0.01 milliliters. Place the semen sample in a 37 degrees Celsius environment for 15 to 30 minutes, and aspirate it into a wide-bore plastic disposable pipette. If the semen does not liquefy within 60 minutes, add an equal amount of fertilization medium. Repeatedly inhale and exhale the mixture using a disposable plastic transfer pipette until it liquefies completely. Measure the pH value of the semen using precision pH test paper with a range of six to 10. Take 10 microliters of fully liquified semen and drip it onto a macular counting chamber. Evaluate sperm motility under a 200X field using an optical microscope. Add five microliters of semen to the fertilization medium microdroplet at the left starting point of the U-shaped track in the ICSI operating dish. Adjust the angle and position of the oocyte-holding needle and injection needle under the 4X objective lens. After verifying the setup, transfer the selected oocyte into the oocyte processing medium microdroplet in the ICSI operating dish. Place the operating dish containing the oocytes onto the hot stage of the prepared microscope operator. After adjusting the focus, lower the injection needle into the PVP strip of the ICSI operating dish. Move the injection needle into the long strip on the right side of the U-shaped track and extract high quality spermatozoa. Transfer the sperm into the PVP strip on the right side and place the sperm at the bottom of the operating dish. Gently press the injection needle in the middle or lower section of the sperm tail. Quickly pull the injection needle back and scratch the sperm to immobilize it. Now, inhale the sperm from the tail to the head into the injection needle. Slowly inject sperm into the cytoplasm of the oocyte, and incubate the injected oocyte. Add 1.5 milliliters of 45% density gradient centrifugation solution to a 15 milliliter sterile conical bottom test tube. And slowly add 1.5 milliliters of 90% density gradient centrifugation solution to the bottom of the 45% gradient solution. Now, add the liquified semen onto the gradient centrifugation solution, and spin the tube at 300 G for 15 minutes at room temperature. After removing the supernatant, collect approximately 0.5 milliliters of the remaining sperm sediment and mix it with three milliliters of fertilization medium. Centrifuge the mixture at 200 G for five minutes at room temperature. Carefully pipette out the supernatant, leaving approximately 0.2 milliliters of sediment, then shake and mix to resuspend the sediment. Finally, count the sperm concentration and motility before storing the sample in 37 degrees Celsius, 6% carbon dioxide incubator for later use. Place a tube containing 25 microliters of low melting point agarose solution into a 90 to 100 degrees Celsius water bath for one to two minutes. Then transfer the tube to a 37 degrees Celsius water bath for five minutes until the temperature stabilizes. Add three to 10 microliters of semen to low melting point agarose tubes and mix thoroughly. Drop 30 microliters of the mixture onto a pretreated glass slide in a horizontal position. After covering, place the pretreated glass slide in a refrigerator at two to eight degrees Celsius for four minutes, ensuring it remains in a horizontal position. Remove the slide from the refrigerator, and gently slide off the cover glass. Quickly immerse the pretreated glass slide in denaturing solution for seven minutes. Remove the slide and make it stand upright. Then use filter paper to absorb excess water droplets without touching the specimen area. Wash the slide in distilled water for five seconds. Immerse the glass slide serially in the appropriate solutions for the specific period. Then keeping the pretreated glass slide horizontal, add around one milliliter of the mixed dye solution onto the slide. After staining for five minutes, gently rinse the stained glass slide with distilled water 10 to 15 times. High quality sperm with good progressive motility swam smoothly along the edge of the U-shaped track, making it easier for the intracytoplasmic sperm injection needle to grasp them individually. While good sperms were obtained using both the methods, the DNA Fragmentation Index of high quality sperm separated using the density gradient centrifugation method was significantly higher compared to the U-shaped horizontal swimming method.