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

Summary

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

Abstract

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of centrifugation on sperm DNA. The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 µL fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 µL fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 °C with 6% CO₂ to equilibrate. Subsequently, 3 µL of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% ± 3.2%, whereas the DFI in the UHS group was 1.7% ± 1.1%. The difference between the two groups was statistically significant (P < 0.05).

Introduction

Semen optimization and spermatozoa preparation techniques play a crucial role in obtaining cell fractions enriched with structurally and functionally superior spermatozoa, which is a key step in human-assisted reproductive technology¹. The purpose of semen optimization is to: (1) Reduce or remove prostaglandins, immune-active cells, anti-sperm antibodies, immobile low-quality sperm, bacteria, and debris in the seminal plasma; (2) Reduce or eliminate the viscosity of semen; and (3) Promote sperm capacitation and enhance fertilization capability. An ideal sperm preparation technique should recover a highly functional sperm population that preserves DNA integrity and does not induce dysfunction through the production of reactive oxygen species (ROS) by sperm and leukocytes².

The most widely used sperm preparation technology currently is the DGC method. The advantages of this method are its high recovery rate³ and easy standardization. In practical use, it can be flexibly selected based on the quality of the specimen for the double-density gradient method⁴, mini-DGC method, or single-layer gradient centrifugation method⁵. This method can be used to prepare high-quality sperm with good vitality, free from cell debris, contaminated white blood cells, non-germ cells, and degenerate germ cells. However, the disadvantage of this method is that it requires centrifugation, which can cause damage to sperm DNA⁶.

The method presented here was adapted from the original study by Baldini et al.⁷, which focused on horizontal sperm migration in injection dishes. This modified method incorporates a U-shaped horizontal lane to separate high-quality sperm with strong vitality. It avoids DNA damage caused by centrifugation and minimizes the influence of dead sperm, cell debris, and other viscous impurities during intracytoplasmic sperm injection (ICSI) procedures.

The specific approach involves using a fertilization medium to create a UHS lane in the ICSI operating dish. A 10 µL fertilization medium microdroplet is placed at the left starting point of the UHS lane to hold the semen. Two additional 10 µL fertilization medium buffer droplets are positioned at intervals in the left middle section of the UHS lane, and all droplets are connected by fertilization medium. After covering the setup with cultivation oil, the dish is incubated overnight at 37 °C with 6% CO₂ for equilibration. Subsequently, 3 µL of semen is added to the microdroplet at the starting point on the left side of the UHS lane. High-quality sperm swim to the track on the right side of the UHS lane, facilitating their collection with the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities primarily remain at the original location or in the buffer droplets.

The microfluidic chip simulates the natural selection process in the female reproductive tract, enabling the optimal isolation of high-quality sperm from semen without centrifugation. This is critical for improving sperm motility⁸, reducing the sperm DNA fragmentation index⁹, and enhancing pregnancy outcomes¹⁰. However, the fabrication of such devices is complex, costly, and challenging to implement widely.

The protocol described herein offers a novel, simple, and feasible alternative. By leveraging sperm motility characteristics, this method achieves results comparable to those of microfluidic technology. The prepared sperm exhibit strong vitality, low DNA fragmentation indices, and are well-suited for use in ICSI.

Protocol

This study was approved by the Medical Ethics Committee of The Affiliated Huai'an First People's Hospital of Nanjing Medical University (Approval number: KY-2024-181-01). Informed consent was obtained from the patients whose samples were used in this study. The procedure should be conducted by experienced personnel in accordance with good laboratory practices and clinical guidelines^11,12. The details of the reagents and equipment used are provided in the Table of Materials.

1. Preparation of an ICSI operating dish

1. Use 20 µL fertilization medium to create U-shaped tracks in the ICSI operating dish (Figure 1). The length of the left track of the U-shaped track should be about 30 mm, and the length of the right track should also be about 30 mm.
2. Suck 10 µL fertilization medium with a pipette and create a circular droplet at the left starting point of the U-shaped track. Connect it with the left starting point of the U-shaped track to add the semen stock solution.
3. Create two 10 µL fertilization medium buffer droplets at intervals on the left middle section of the UHS lane. Connect all droplets with the fertilization medium.
4. Create a long strip of liquid droplet on the right side of the U-shaped lane using 2.5 µL Polyvinyl pyrrolidone (PVP). Ensure the PVP strip is parallel to the right lane of the U-shaped lane.
5. Make six microdroplets using the oocyte processing medium on the right side of the PVP strip, with 20 µL per droplet.
6. Add 7 mL culture oil to the dish, ensuring that the oil covers the highest point of the droplet. Place it in a 37 °C, 6% CO₂ incubator overnight for balance.

2. Semen sample collection

1. Provide patients with clear written and verbal instructions regarding the collection of the semen sample. Ensure the sample collection is complete, and instruct patients to abstain from ejaculation for 3-7 days prior to collection.
2. Collect semen via masturbation into a disposable, sterile, and non-toxic specialized sperm collection cup. Ensure the entire ejaculate is collected, and instruct the individual to report any loss of any fraction of the sample.
3. Verify the names, valid identification documents, and fingerprints of the patient's spouse. Mark the name and medical record number of the patient's spouse on the body and lid of the sperm collection cup.
4. After the male patient has collected semen, hand over the specimen face-to-face between the staff and the patient. Take a small amount of the semen sample for packaging and storage. Ensure the patient signs at the packaging area.
5. Number and store the encapsulated specimens for 2 years. Record patient information, identification number, and signatures of the recipient and witnesses in the semen processing logbook.
6. If any staff member of the reproductive center discovers a suspicious patient's identity, stop receiving the specimen and re-verify the patient's identity. Do not receive two or more specimens at the same time. If there is suspicion of confusion in the specimens, stop receiving them.

3. Analysis of semen specimen

1. Observe the color of the semen specimens.
   NOTE: Normal semen appears homogeneous and grayish-white after liquefaction. Semen with very low sperm concentration may appear transparent. If red blood cells are present, the semen may appear reddish-brown. If the patient has jaundice or takes certain vitamins, the semen may appear yellow.
2. Measure the volume of semen using the weighing method, accurate to 0.1 mL.
3. Place the semen sample at 37 °C for 15-30 min. Aspirate it into a wide-bore (approximately 1.5 mm diameter) plastic disposable pipette, allowing the semen to drop by gravity. Observe the length of any thread.
   NOTE: A normal liquefied ejaculate forms small, discrete drops. If the viscosity is abnormal, the drop will form a thread longer than 2 cm.
4. If the semen still does not liquefy within 60 min, add an equal amount of fertilization medium. Then, repeatedly inhale and exhale the mixture using a disposable plastic transfer pipette until it liquefies completely.
5. Measure the pH value of the semen using precision pH test paper (pH 6.0-10.0).
6. Take 10 µL of fully liquefied semen and drip it onto a Makler counting chamber. Evaluate sperm motility under a 200x field with an optical microscope. Observe the presence of sperm agglutination and non-sperm cells.
7. Evaluate sperm concentration under a 200x field with an optical microscope.

4. Picking up oocytes

1. Prepare a dish for picking up oocytes (OPD) the day before oocyte retrieval. Add 2.5 mL of oocyte processing medium to a 35 mm sterile culture dish. Then, add 1.5 mL of oil and place the dish in a 37 °C incubator overnight for equilibration.
2. Prepare two dishes for oocyte culture (OCD) the day before oocyte retrieval. Add 1 mL of fertilization medium to the inner ring of a sterile central well culture dish, then add 1 mL of oil. Add 4 mL of fertilization medium to the outer ring. Place the dish in a 37 °C, 6% CO₂ incubator to equilibrate overnight.
3. Turn on all heaters and heating plates and measure the temperature 30 min before oocyte retrieval.
4. Wash hands with hand sanitizer and rinse thoroughly with running water. Wear clean, sterile, and dust-free gloves.
5. Preheat the sterile culture dish on a 37 °C heating platform.
6. Strictly verify the patient's identity before oocyte retrieval.
7. Inhale follicular fluid into sterile test tubes through negative pressure, then immediately hand it over to laboratory embryologists.
8. Pour all follicular fluid into sterile culture dishes and search for oocyte corona cumulus complexes (OCCCs) under a low magnification stereomicroscope. Pick up the OCCCs using a pipette and place them in an OPD for temporary storage at 37 °C.
9. Record the amount, color, number of follicles, and number of OCCCs in the follicular fluid.
10. After oocyte retrieval, transfer the OCCCs to OCD using a sterile Pasteur pipette. Quickly place them in a culture incubator (37 °C, 6% CO₂, 5% O₂, and saturated humidity) for 2 h.
11. Prepare a four-well dish, add 0.5 mL of hyaluronidase solution to the first well, and 1 mL of oocyte processing medium to each of the remaining three wells. Incubate at 37 °C for 1 h.
12. Use a Pasteur pipette to transfer OCCCs from step 4.10 into a well-containing hyaluronidase for 30 s. Then, transfer the OCCCs into a well-containing oocyte processing medium.
13. Use a 150 µm diameter oocyte stripping tube to blow and aspirate the OCCCs to remove granulosa cells. Then, use a new Pasteur pipette to transfer the oocytes into the oocyte processing medium for washing. Transfer them to another preequilibrated OCD and place them in a 37 °C, 6% CO₂ incubator.

5. Sperm selection and ICSI operation

1. Add 5 µL of semen to the fertilization medium microdroplet at the left starting point of the U-shaped track in the ICSI operating dish, which was prepared from step 1.1 to step 1.6.
2. Place the ICSI operating dish into an incubator at 37 °C, 6% CO₂ for 30-60 min.
3. Open the inverted microscope, microscope operating system, and stage heating stage to ensure that all operational controls are restored to their original controllable range, allowing for smooth and comfortable operation.
4. Install the ICSI needle into the needle holder. Fix the needle holder on the micromanipulator, adjust the holding needle and injection needle in the 4x objective lens, and sequentially adjust their angles and positions so that the two needles are relative and parallel to the stage. Check the range of movement of the operating needle under the × 20 objective lens.
5. Adjust the angle and position of the oocyte holding needle and injection needle under the 4x objective lens, so that the two needle heads are relative and parallel to the stage. Move the operating needle forward, backward, left, and right. Check the movement range of the operating needle under the 10x and 20x objectives.
6. Raise the oocyte holding needle and injection needle to ensure that the height between the operating table and the heating table allows for easy placement of the operating vessel without touching the operating needle.
7. After double-checking, transfer the selected oocytes from step 4.12 into oocyte processing medium microdroplets in the ICSI operating dish, one per droplet.
8. Place the operating dish containing the oocyte on the hot stage of the prepared microscope operator.
9. Adjust the focal length of the microscope under the 10x objective lens to make the edges of the microdroplets inside the operating dish clear and visible.
10. Lower the injection needle into the PVP strip of the ICSI operating dish. Adjust the microscope to make the injection needle clearly visible, and inhale a small amount of PVP into the injection needle at the same time.
11. Move the injection needle into the long strip on the right side of the U-shaped track and extract high-quality spermatozoa with good morphology and progressive motility.
12. Transfer the sperm into the PVP strip on the right side and place the sperm at the bottom of the operating dish.
13. 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 make it stop.
14. Inhale the sperm from the tail to the head into the injection needle.
15. Transfer the injection needle into the oocyte processing medium droplet containing the oocyte on the right side.
16. Lower the oocyte holding needle and gently move the oocyte to position the first polar body at 12 o'clock, securing the oocyte.
17. Adjust the microsurgical needle and oocyte membrane to the same horizontal plane. Push the sperm to the injection needle tip.
18. Vertically pass through the zona pellucida at 3 o'clock on the oocyte and continue to inject the needle until it reaches the center of the oocyte or slightly crosses the center position, with a slight retraction of the injection needle.
    NOTE: When rapid reflux occurs in the cytoplasm and sperm, it indicates that the oocyte membrane has broken and aspiration has stopped.
19. Slowly inject sperm into the cytoplasm of the oocyte and exit the injection needle. The depth of sperm injection into the cytoplasm should be about 50%-75% of the oocyte diameter.
20. After withdrawing the injection needle, adjust the negative pressure of the oocyte holding needle to release the oocytes.
21. Repeat the above steps until all mature oocytes have been injected.
22. Transfer the ICSI dish from the microscope hot plate to the dissecting microscope.
23. Remove the cleavage culture dish from the incubator.
24. Transfer the injected sperm oocyte to a microdroplet in a cleavage culture dish.
25. Place the cleavage culture dish back into a 37 °C, 6% CO₂, 5% O₂ incubator.

6. Preparation of sperm using DGC method

1. Add 1.5 mL of 45% density gradient centrifugation solution to a 15 mL sterile conical bottom test tube.
2. Slowly add 1.5 mL of 90% density gradient centrifugation solution to the bottom of the 45% density gradient centrifugation solution, maintaining the interface between the two liquids.
3. Gently add the liquefied semen onto the gradient centrifugation solution and centrifuge at 300 x g for 15 min (at room temperature).
4. Remove the supernatant and add approximately 0.5 mL of the remaining sperm sediment to 3 mL of fertilization medium. Blow and mix well.
5. Centrifuge at room temperature at 200 x g for 5 min.
6. Pipette the supernatant and leave about 0.2 mL of sediment.
7. Add an appropriate amount of fertilization medium to resuspend the sediment, count sperm concentration and vitality, and record. Place in a 37°C, 6% CO2 incubator for later use.

7. Detection of sperm nuclear DNA integrity (Sperm Chromatin Dispersion Method, SCD)

1. Adjust the indoor temperature to 20-28 °C before conducting the experiment. Remove the reagent kit and let it equilibrate at room temperature for 30-60 min.
2. Prepare the denaturing solution: Take 0.8 mL of concentrated denaturing solution and add it to 100 mL of distilled water.
3. Prepare the 70% ethanol solution: Take 26.65 mL of distilled water and 70.35 mL of anhydrous ethanol to prepare the 70% ethanol solution.
4. Prepare the 90% ethanol solution: Take 9.55 mL of distilled water and 90.45 mL of anhydrous ethanol to prepare the 90% ethanol solution.
5. Place the low melting point agarose tube (containing 25 µL of low melting point agarose solution) into a 90-100 °C water bath for 1-2 min until the agarose gel is completely melted. Then, place the tube in a 37 °C water bath for 5 min until the temperature is constant.
6. Take 3-10 µL of semen before optimization treatment and sperm suspension after optimization treatment. Add them to different low melting point agarose tubes, then mix thoroughly.
7. Take 30 µL of the low melting point agarose suspension containing sperm and drop it onto a pre-treated glass slide in a horizontal position.
8. Gently cover the cover glass (22 mm x 11 mm in size) with the slide, avoiding the formation of bubbles as much as possible.
9. Place the pre-treated glass slide in a refrigerator at 2-8 °C for 4 min, keeping the slide in a horizontal position throughout the entire process. Remove the glass slide from the refrigerator and gently slide to remove the cover glass.
10. Quickly immerse the pre-treated glass slide in the denaturing solution for 7 min.
11. Remove the pre-treated glass slide and soak it in distilled water for 5 s.
12. Remove the pre-treated glass slide and make it stand upright. Use filter paper to absorb any water droplets on the surface of the slide, and avoid touching the specimen area.
13. Immerse the pre-treated glass slide in the lysis buffer and react accurately for 20 min.
14. Immerse the pre-treated fragments in the washing solution for 3 min to wash away the lysis solution.
15. Remove the pre-treated glass slide and immerse it in a 70% ethanol solution for 2 min.
16. Remove the pre-treated glass slide and immerse it in a 90% ethanol solution for 2 min.
17. Remove the pre-treated glass slide and immerse it in anhydrous ethanol solution for 2 min.
18. Remove the pre-treated glass slide and let it air dry naturally.
19. Prepare the Wright stain solution A and solution B in a brown empty bottle in a 1:1 ratio.
20. Place the pre-treated glass slide horizontally and add the mixed dye solution onto the slide, ensuring the dye solution covers the entire slide (about 0.5-1 mL of dye solution).
21. After staining for 5 min, gently rinse the stained glass slide with distilled water 10-15 times to remove excess staining agent.
22. Let the glass slide dry naturally and observe it under an optical microscope at 400×. Count more than 200 sperm and determine the percentage of abnormal sperm with small halo rings, no halo rings, and degeneration.

8. Statistical analysis

1. Perform statistical analysis using commercially available software.
2. Compare the DFI (%) between original semen, DGC, and UHS using the Friedman test.
   NOTE: For pairwise comparisons of two methods, use the Wilcoxon signed-rank test. Consider a p-value ≤ 0.05 as statistically significant.

Results

The UHS and DGC methods were used to optimize the treatment of 21 samples and compare the sperm DNA fragmentation index between the two methods. The method using the UHS track in the ICSI dish can replace DGC, which may cause damage to sperm. High-quality sperm with good progressive motility swim smoothly along the edge of the U-shaped track (Figure 2), making it easier for the ICSI needle to grasp them individually. The high-quality sperm isolated using the UHS track have lower DFI (

Discussion

The key step in separating high-quality sperm using the UHS method described in this article is the establishment of a UHS lane with buffer droplets. Both the UHS lane and the buffer droplets are created using washed and received semen. The UHS lane guides high-quality sperm to swim freely and accumulate along the edge of the track, making it easy to collect. The buffer droplets reduce the viscosity of semen, filtering out dead sperm, cell debris, and other impurities. The distance between the right side of the UHS lane ...

Materials

Name	Company	Catalog Number	Comments
7% Polyvinyl pyrrolidone Solution	Vitrolife Sweden AB	10111	ICSI
Aspirator	LABOTECT	-	Aspirator
Biological clean workbench	Suzhou Antai	-	Biological clean workbench
Blastocyst culture medium	Vitrolife Sweden AB	10132	Blastocyst culture medium
Cleavage culture medium	Vitrolife Sweden AB	10128	Cleavage culture medium
CO2 incubator	Thermo Scientific	-	CO2 incubator
Culture oil	Vitrolife Sweden AB	10029	OVOIL
Disposable plastic transfer pipette	BD Falcon	357575	disposable plastic transfer pipette
Fertilization medium	Vitrolife Sweden AB	10136	G-IVF PLUS
ICSI operating dish	BD Falcon	351006	Petri dish
Instant hyaluronidase	Vitrolife Sweden AB	10017	Instant hyaluronidase
Inverted microscope	NIKON	-	Inverted microscope
IVF Workstation	Denmark K-SYSTEM	-	IVF Workstation
Makler counting chamber	Sefi Medical Instruments		Makler counting chamber
Micro operating system	NIKON	-	Micro operating system
Oocyte processing medium	Vitrolife Sweden AB	10130	G-MOPS PLUS
Optical microscope	OLYMPUS	-	Optical microscope
Phase contrast microscope	NIKON	-	Phase contrast microscope
Sperm Counting Board	Markler	-	Sperm Counting Board
Sperm gradient separation solution	Vitrolife Sweden AB	10138	SpermGrad
Sperm nucleus DNA integrity Kit	Shenzhen HuaKang	-	Sperm Nucleus DNA Integrity Kit (SCD)
Stereoscopic microscope	NIKON	-	Stereoscopic microscope
Tabletop centrifuge	HETTICH	-	Tabletop centrifuge
Thermostatic test tube rack	GRANT	-	Thermostatic test tube rack
Tri-gas incubator	ASTEC	-	Tri-gas incubator