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Intérêt de l’étude de l’oxydation de l’ADN des spermatozoïdes par marquage de la 8-oxo-guanine en cytométrie en flux chez l’homme infertile

Detection of oxidative DNA damage by flow cytometry and its association with male infertility

Résumé

Dans le sperme, le stress oxydatif est essentiellement du à une production excessive de dérivés actifs de l’oxygène (DAO) d’origine essentiellement leucocytaire. L’oxydation de l’ADN estdue à l’action directe des DAO qui produisent en conséquence plusieurs adduits dont le plus étudié est la 8-oxo-guanine. L’intégrité de l’ADN est essentielle pour la fécondance du spermatozoïde et elle constitue de nos jours un sujet d’intérêt pour les chercheurs et cliniciens dans le monde. Si les moyens d’évaluation de l’intégrité globale de l’ADN spermatique se sont bien développés ces dernières années, les tests d’évaluation du dommage de l’ADN d’origine oxydative sont peu documentés.

L’objectif de ce travail est de faire le point sur les différents tests d’étude de l’intégrité de l’ADN spermatique couramment utilisés selon la littérature, et de présenter les résultats de notre étude sur l’oxydation de l’ADN par marquage de la 8-oxo-guanine en cytométrie en flux chez l’homme infertile.

Notre travail a porté sur 15 échantillons de sperme qui ont fait l’objet d’une analyse spermiologique selon les recommandations de l’OMS, avec une mesure de la concentration des leucocytes par la méthode cytochimique révélant la Peroxydase dans les granulations cytoplasmiques. L’étude de l’oxydation de l’ADN a été réalisée grâce à un kit de marquage de la 8-oxo-guanine en cytométrie en flux.

Nous avons montré par l’étude de la régression linéaire une forte corrélation entre l’oxydation de l’ADN et le taux de leucocytes dans le sperme (p=0,006, ß=0,7). Un seuil de leucocytes de 250 000/ml de sperme était associé à une augmentation significative de l’oxydation de l’ADN (p=0,03).

Nous concluons que la 8-oxo-guanine pourrait être considérée comme un biomarqueur de l’action directe du stress oxydatif sur l’ADN spermatique qui semble être vulnérable à des taux relativement faibles de DAO produits par des leucocytes présents à une concentration largement inférieure au seuil de la leucospermie défini par l’OMS.

Abstract

Oxidative stress in semen is essentially due to excessive production of oxygen-reactive species (ORS) essentially derived from leukocytes. DNA oxidation is due to the direct action of ORS which produce several adducts the most extensively studied of which is 8-oxo-guanine. Integrity of DNA is essential for the fertility of sperm and is an important subject of research for scientists and clinicians all over the world. Although evaluation of the global integrity of sperm DNA has considerably developed over recent years, few tests are available to document oxidative DNA damage.

This study was designed to review the various tests of sperm DNA integrity commonly used in the literature and to present the results of our study on DNA oxidation with 8-oxo-guanine labelling by flow cytometry in infertile men.

This study was based on 15 semen samples that were submitted to sperm analysis according to WHO guidelines, with determination of the leukocyte concentration by a cytochemical method revealing peroxidase in cytoplasmic granulations. The DNA oxidation study was performed with 8-oxo-guanine labelling by flow cytometry.

Linear regression analysis showed a strong correlation between DNA oxidation and leukocyte count in the semen (p = 0.006, r = 0.7). A leukocyte cut-off of 250,000/ml of semen was associated with a significant increase of DNA oxidation (p = 0.03).

8-oxo-guanine can therefore be considered to be a biological marker of the direct action of oxidative stress on sperm DNA which appears to be susceptible to relatively low levels of ORS produced by leukocytes present at concentrations well below the limit of leukospermia defined by WHO.

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Correspondence to Nozha Chakroun Feki or Nassira Zribi or Henda Eleuch or Radouane Gdoura or Afifa Sellami or Ali Bahloul or Adnene Hammami or Jalel Gargouri or Tarek Rebai or Leila Keskes Ammar.

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Chakroun Feki, N., Zribi, N., Eleuch, H. et al. Intérêt de l’étude de l’oxydation de l’ADN des spermatozoïdes par marquage de la 8-oxo-guanine en cytométrie en flux chez l’homme infertile. Androl. 18, 197–205 (2008). https://doi.org/10.1007/BF03040755

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Mots clés

  • leucospermie
  • stress oxydatif
  • dommage de l’ADN
  • 8-oxo-guanine
  • spermatozoïde
  • infertilité

Key words

  • DNA oxidative damage
  • leukocyte
  • 8-oxo-guanine
  • sperm
  • oxidative stress
  • infertility