# DNA Fragmentation Analysis in Human Sperm—Technical Instructions to Prevent False Positives and Negatives in Angle-Modulated Two-Dimensional Single-Cell Pulsed-Field Gel Electrophoresis

## TL;DR

This paper provides technical guidelines to improve the accuracy of DNA fragmentation analysis in human sperm using advanced gel electrophoresis methods.

## Contribution

The paper introduces an anti-ROS SCPFGE system and outlines procedures to prevent false results in DNA fragmentation analysis.

## Key findings

- Conventional DNA fragmentation methods have technical limitations that can lead to false positives or negatives.
- Angle-modulated 2D-SCPFGE is the most sensitive method for analyzing single-nuclear DNA fibers.
- Low-temperature storage is recommended to prevent artifactual DNA cleavage from ice crystals.

## Abstract

Over the past two decades, numerous studies have examined the etiological significance of DNA fragmentation in human sperm using methods such as the comet assay (CA), the sperm chromatin structure assay, the sperm chromatin dispersion assay, and the TUNEL assay. We developed single-cell pulsed-field gel electrophoresis techniques, including one-dimensional (1D-SCPFGE) and angle-modulated two-dimensional (2D-SCPFGE), to detect early signs of naturally occurring DNA fragmentation. Comparative studies using purified human sperm with and without DNA fragmentation revealed some technical limitations in the conventional methods. This technical review outlines the procedures to ensure the quantitative performance of SCPFGE: (1) The mass of naked DNA was prepared through simultaneous in-gel swelling and proteolysis, which are highly sensitive to chemical and physical factors. Notably, these processes are vulnerable to reactive oxygen species (ROS). We developed the anti-ROS SCPFGE system to prevent artifactual cleavages. (2) 1D-SCPFGE discharges long-chain fibers from the origin, separating fibrous and granular segments beyond the tips of the fibers. (3) During continuous electrophoresis after 150° rotation (2D-SCPFGE-0-150), long-chain fibers unexpectedly extended diagonally backward from the origin, with long fibrous segments pulled out from a bundle that extended during the first electrophoresis, indicating some fibrous segments were embedded within the long-chain fibers. Even when SCPFGE was employed, one-directional current led to false negatives. (4) 2D-SCPFGE with angle rotation is currently the most sensitive imaging method for single-nuclear DNA fibers. However, without knowing the size of DNA fragments, it remains a semi-quantitative analysis. (5) To prevent artifactual DNA cleavage caused by ice crystals, low-temperature liquid storage is recommended. (6) The in-gel proteolyzed naked DNA is suitable as a substrate for chemical and enzymatic DNA cleavage analyses.