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-strand and double-strand breaks, etc. Moreover, damaged sperm DNA can impair male fertility and adversely affect pregnancy outcomes in assisted reproductive technology (ART)⁴, while also increasing the risk of recurrent pregnancy loss⁵. In 2021, the European Association of Urology (EAU) recommended the inclusion of DFI testing in the male fertility assessment system⁶.

At present, the integrity of sperm DNA is mainly evaluated by detecting the sperm DNA fragmentation index (DFI). The sperm chromatin structure assay (SCSA) by flow cytometry is the primary method for assessing DFI⁷. Two flow cytometry gating methods for sperm DFI detection have been reported in the literature. One is the polygonal gating method (PGG), which can simultaneously report two key indicators, such as sperm DFI and HDS⁸. PGG is a classic SCSA approach characterized by a smaller detection coefficient of variation and superior repeatability⁹. However, it cannot distinguish the severity of DNA damage. Another type is the cruciform gating method (CFG), which can report severe damage marker of sperm DNA (DFIs), mild damage marker of sperm DNA (DFIm), and the total DFI of the cruciform gating method (DFIc). DFIc is equal to the sum of DFIs and DFIm. Compared with current technologies for assessing sperm DNA integrity, the CFG method can reflect the severity of sperm DNA damage¹⁰.

To determine which method is more suitable for assessing sperm quality, this study employed both the PGG method and the CFG method to measure DFI and related parameters in sperm. The correlation between the results of these two methods and conventional semen analysis parameters was compared. To our best knowledge, this is the first study to analyze the correlation of detection results between the two methodologies, PGG and CFG.

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 chromatin of sperm with undamaged DNA retains its intact double - stranded structure. The fluorescent dye acridine orange (AO) binds to single - stranded DNA and emits red fluorescence when excited by a 488 nm laser. When bound to double - stranded DNA, it emits green fluorescence when excited by a 488 nm laser. Based on the number of red and green fluorescent signals captured by the flow cytometer, the ratio of red fluorescence to the total of red and green fluorescence is calculated, which represents the sperm DFI.

The CFG settings were implemented according to the overview provided by Yang et al.¹⁰. The flow cytometer should be configured using the lowest green fluorescence and the highest red fluorescence measured in AO-stained normal sperm as boundary references (Figure 2). The following is a list of the cellular characteristics of each quadrant in the CFG:
Quadrant Q1 is indicative of normal sperm. It was observed that the sperm DNA was intact, and the binding amount of AO was minimal, resulting in green fluorescence.
Sperms in quadrant Q2 have partially fragmented DNA and appear orange under fluorescence microscopy. Such DNA may be repairable, these sperms are classified as having mild DNA fragmentation (DFIm)¹⁰.
Quadrant Q3 is defined as the non-specific fluorescence that has undergone subtraction.
Sperms in quadrant Q4 have fragmented DNA and appear red under fluorescence microscopy, indicating severe DNA fragmentation (DFIs).

It is proposed that sperms in quadrants Q2 and Q4 both contain fragmented DNA, and their sum is defined as DFIc.

Settings of the PGG for the flow cytometer
The polygonal gate for flow cytometric analysis was established following the method described by Evenson et al.⁸. As shown in Figure 3, the cellular characteristics associated with each gate are as follows:
P3 is indicative of the percentage of cells with denatured DNA.
P4 represents sperm with high DNA stainability (HDS). This group of sperm lacks the normal histone-to-protamine exchange.
The predominant cell populations located outside the P3 and P4 gates consist of normal spermatozoa, exhibiting reduced red fluorescence and predominantly emitting green fluorescence.

Semen sample collection and analysis
All 121 semen samples were from the men who visited the Reproductive Center of Huai'an First Hospital Affiliated to Nanjing Medical University. Semen samples were collected by masturbating after a period of 2-7 days of abstinence. Routine semen analysis was strictly conducted in accordance with the requirements of the WHO Laboratory Manual for the Examination and Processing of Human Semen^11,12. Sperm concentration and motility analyses were carried out using a computer-aided sperm analysis system (CASA). Each sample was analyzed twice. The results were reported as the mean only when the difference between the two results was within the 95% confidence interval; otherwise, resampling and analysis were conducted.

DFI analysis
Calculate the required sample volume for the semen sample so that the final concentration of sperm in reagent A from the flow cytometry kit is 2–3 x 10⁶/mL. Calculation method: Assuming the sperm concentration in the original semen specimen is M x 10⁶/mL, then the required semen volume (µL) = 150/M. Add the calculated semen volume to a flow cytometer tube, then add 50 µL of reagent A and mix gently. Add 100 µL of reagent B, mix gently, and incubate for 30 s at room temperature. Immediately add 300 µL of reagent C and mix gently. Detect the sample using CFG and PGG on the flow cytometer, and analyze at least 5000 spermatozoa.

Statistical analysis
Data were subjected to statistical analysis using GraphPad Prism 6.0. Bland-Altman plots were constructed to assess the level of agreement between PGG and CFG by calculating the mean and differences. The differences between the two methods were plotted against their average values. Furthermore, the Pearson method was utilized to analyze the correlation between PGG and CFG. It was determined that a p-value less than 0.05 was statistically significant.

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	-