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J Reprod Infertil. 2019 Apr-Jun;20(2):95-101.

Evaluation of Sperm Chromatin Integrity Using Aniline Blue and Toluidine Blue Staining in Infertile and Normozoospermic Men.

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Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Gametogenesis Research Center, Fertility, and Infertility Center, Kashan University of Medical Sciences, Kashan, Iran.
Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.



Male infertility is defined as a man lost his ability to fertilize a fertile female naturally. Diagnosis of male infertility cannot be made just according to basic semen analysis. It is necessity to have specific tests for evaluation of chromatin integrity. In this study, an attempt was made to evaluate the sperm chromatin quality in fertile men and infertile subgroup.


Among 1386 couples, 342 men were categorized into normospermia and 1044 were infertile and they were referred to Yazd Research and Clinical Center for infertility treatment. Standard semen analysis and sperm nuclear maturity tests including aniline blue (AB) and toluidine blue (TB) staining were done. Data were analyzed by SPSS software. The p≤0.05 was considered statistically significant.


The mean value of TB staining was significantly higher in infertile group compared to normospermic group (p=0.005). Mean of sperm normal morphology was lower in idiopathic infertile men in comparison with normozoospermic men (p= 0.001). The highest negative correlation was obtained between sperm count and AB staining. Progressive motility was negatively correlated with AB and TB staining in both groups but there was no significant difference between AB staining and progressive motility in men normospermia group.


Sperm chromatin staining using AB and TB showed a negative association between sperm chromatin condensation with sperm count, normal morphology and progressive motility. It seems that the AB and TB test may be useful for the assessment of male fertility potential.


Aniline blue; Infertility; Sperm; Toluidine blue


Conflict of interest statement

Conflict of Interest The authors have no financial or nonfinancial conflicts of interest.

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