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Updated: Aug 6, 2026

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects
Published on: April 21, 2022
High frequency of sperm function defects in normozoospermic infertile men
Vertika Singh1, Arijit Chakraborty1,2, Sameer Trivedi3
1Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi, India.
Objectives:
Conventional semen analysis often fails to detect hidden functional defects contributing to male infertility. Factors such as sperm DNA-integrity, oxidative stress, MMP, and acrosome integrity are critical for fertilization and early embryonic development. This study aimed to evaluate sperm function defects in normozoospermic infertile men and highlight limitations of routine semen parameters in infertility assessment.
Methods:
Semen samples underwent standard semen analysis. Sperm DNA damage was assessed using Acridine Orange staining, acrosome integrity by FITC-PSA staining, and ROS levels along with MMP were measured using flow cytometry. Appropriate statistical analyses were performed to compare infertile men and fertile controls and determine correlations among functional parameters.
Results:
Among clinically screened infertile patients, 23 % were normozoospermic according to WHO 2010 criteria. Sperm function parameters were evaluated in normozoospermic infertile men (n=45) and fertile controls (n=17). Infertile men exhibited significantly higher ROS levels and DNA damage (p<0.001), alongside reduced acrosome integrity and MMP (p<0.001) compared to controls. ROS showed a positive correlation with DNA damage and acrosome-reacted sperm, and a negative correlation with MMP.
Conclusions:
Functional sperm defects may underlie infertility in a substantial proportion of normozoospermic men. Incorporating sperm function tests as second-line investigations may improve diagnosis and management of unexplained male infertility.
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