Comparison of Sperm DNA Analysis Outcomes Using Different Gating Strategies in Flow Cytometry

Summary

Polygonal gating (PGG) and cruciform gating (CFG) are flow cytometry methods for detecting sperm DNA integrity. PGG measures the DNA fragmentation index (DFI) and the high DNA staining index (HDS). CFG reports severe DFI, mild DFI, and overall DFI. This study aims to compare the results and correlations between them.

Abstract

Sperm DNA integrity is a key factor in ensuring successful fertilization, embryo development, and ongoing pregnancy. Sperm chromatin structure assessment (SCSA) using flow cytometry after DNA denaturation and acridine orange staining is considered the gold-standard method for evaluating sperm DNA. Two gating strategies for flow cytometry–based assessment of the sperm DNA fragmentation index (DFI) have been reported in the literature: polygonal gating (PGG) and cruciform gating (CFG). The aim of the present study was to compare the relationships of assay results between PGG and CFG, and to analyze their correlations with parameters of basic semen analysis. Data obtained independently by the two methods were compared using statistical analysis, including Bland-Altman plots and regression analysis. A total of 121 male outpatients undergoing fertility assessment at our hospital's reproductive center were selected via a completely random sampling method. Sperm DFI and related indicators were detected by flow cytometry using PGG and CFG, respectively. The results demonstrated that DFI values obtained from the two methods showed good agreement. The correlation coefficients between DFIp from the PGG method and DFIm, DFIs, and DFIc from the CFG method were 0.6497, 0.9404, and 0.9667, respectively (All p < 0.01). High DNA staining index (HDS) showed a slight negative correlation with DFIs (r = -0.3042, p < 0.05). The percentage of progressively motile sperm was negatively correlated with DFIp, DFIs, and DFIc (r = -0.3824, -0.3794, -0.3574, respectively, all p < 0.05). No significant correlation was observed between HDS and semen volume, sperm concentration, total sperm count, or PR (%). These findings indicate that DFIs are more closely associated with male fertility, suggesting that the CFG method, which provides this parameter, may be more suitable for clinical assessment of sperm DNA damage.

Introduction

Sperm DNA integrity refers to the integrity and functional stability of the DNA molecular structure in the sperm nucleus. Specifically, it is characterized by normal chromatin conformation, intact base sequences without breakage or oxidative damage, and the capacity to accurately transmit genetic information to offspring. During spermatogenesis, when oxidative stress increases¹, chromatin remodeling is abnormal², or the sperm is affected by environmental toxins³, the integrity of sperm DNA will be damaged, resulting in base mismatch, loss, modification, DNA addition and cross-linking, single-stran....

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. Overall schematic diagram of the workflow presented in Figure 1. The details of the reagents and equipment utilised are outlined in the Table of Materials.

Settings of CFG for the flow cytometer
After acid treatment, the nuclear chromatin of sperm with damaged DNA shows single - stranded DNA, while the nuclear ....

Results

Among the 121 study participants (mean age: 32.5 ± 4.9 years; range: 26–44 years), 28 were diagnosed with asthenozoospermia and 4 with oligozoospermia. Baseline demographic characteristics and semen analysis parameters are summarized in Table 1.

As demonstrated in Figure 4, the Bland-Altman plot revealed that the sperm DFI results obtained by the PGG and CFG methods exhibited excellent consistency (r = 0.9667, p = 0.0000). Only two samples showed .......

Discussion

The 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen¹² recommends four methods for assessing sperm DNA integrity: terminal deoxynucleotidyl transferase (dUTP) nick end labelling (TUNEL), single cell gel electrophoresis (Comet) assays, SCSA, and sperm chromatin dispersion (SCD) test. Among these, SCSA has been widely adopted in andrology laboratories due to its use of flow cytometry for analysis, which offers high throughput, efficiency, and objectivity, as.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Computer-assisted sperm analysis system	Beijing Suijia Software Co., Ltd	SA-II
Flow cytometer	Shenzhen Mindray Bio-Medical Electronics Co., Ltd	Cyto E6
Flow cytometry kit	Shenzhen BRED Biotechnology Co., LTD
Makler counting chamber	Sefi Medical Instruments
Sperm nucleus DNA integrity Kit	Shenzhen BRED Biotechnology Co., LTD	-