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 preserv

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

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