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Comment identifier le spermatozoïde vivant?

How to identify living spermatozoa?

Resume

Les spermatozoïdes testiculaires ont une mobilité souvent diminuée voire nulle qui est considérablement altérée par le processus de congélation-décongélation. La difficulté, face à des spermatozoïdes immobiles, est de faire la distinction entre un spermatozoïde vivant et un spermatozoïde mort. La plupart des test de vitalité spermatique explore l’intégrité fonctionnelle et structurale de la membrane plasmique, comme les tests d’exclusion des colorants vitaux ou le test de gonflement hypo-osmotique.

La mobilité spermatique est le seul paramètre qui permet d’avoir la certitude de la vitalité spermatique. Nous avons évalué les effets de la Pentoxifylline (PTX), inhibiteur de la phosphodiestérase de l’AMPc, sur les spermatozoïdes testiculaires décongelés (n=14), afin de démontrer que ce stimulant chimique est capable d’induire ou d’augmenter leur mobilité. La PTX augmente significativement le nombre de spermatozoïdes testiculaires mobiles après décongélation dans les prélèvements de patients présentant une azoospermie sécrétoire (n=8) ou excrétoire (n=6). L’action maximale est atteinte entre 60 et 120 minutes. L’effet de la PTX sur la mobilité est supérieure lorsque les spermatozoïdes sont issus de sujets présentant une azoospermie excrétoire. L’association PTX-gradient de migration amplifie cet effet sur la mobilité dans les prélèvements d’origine excrétoire. Cet effet cumulatif n’est pas observé sur les spermatozoïdes des azoospermies sécrétoires.

Sur les 100 cycles d’ICSI réalisés à l’aide de spermatozoïdes testiculaires ou épididymaires décongelés et traités par la PTX à 3,5mM, aucune différence significative n’a été observée entre les taux de fécondation, le nombre d’embryons obtenus, transférés ou congelés ainsi que les taux de grossesse entre les cycles d’ICSI utilisant des spermatozoïdes testiculaires des azoospermies sécrétoires et ceux des azoospermies excrétoires.

Le Pentoxifylline, en induisant et augmentant la mobilité des spermatozoïdes testiculaires décongelés, facilite la sélection des spermatozoïdes vivants en vue de leur utilisation ultérieure.

Abstract

Testicular sperm extraction (TESE) has been used to retrieve spermatozoa in patients with secretory azoospermia for intracytoplasmic sperm injection (ICSI). However, testicular spermatozoa have poor motility that significantly decreases after cryopreservation and thawing. The major difficulty with testicular spermatozoa is to distinguish between living and dead spermatozoa, as most spermatozoa are immotile. The aim of this study was firstly to report the various methods used to explore spermatozoa vitality. Most tests assess the functional and structural integrity of the sperm membrane, such as staining methods and hypo-osmotic swelling test (HOS-test). We then evaluates the potential of pentoxifylline (PTX), a phosphodiesterase inhibitor of the methylxanthine group, to improve the distinction between living and dead immotile testicular spermatozoa by increasing the number of post-thawed motile spermatozoa. We also analysed the results of 100 ICSI cycles performed with frozen-thawed testicular (n=72) and epididymal (n=28) spermatozoa treated with 3.5 mM PTX.

To test the effect of PTX on motility, 14 samples of frozen-thawed testicular spermatozoa from eight patients with secretory azoospermia and six patients with excretory azoospermia were divided into three equal samples: one sample treated with 3.5 mM PTX, one sample initially migrated on two-layer Percoll gradient and then divided into two aliquots (one treated with 3.5 mM PTX, one without treatment), and the last sample without migration and without PTX treatment. The number of motile spermatozoa was evaluated in 10 μL of each sample with an inverted microscope at 15, 30, 60, 120 minutes and 24 hours. We also compared the outcome of ICSI in 100 cycles using frozen-thawed epididymal or testicular spermatozoa between secretory and excretory patients.

PTX significantly increased the number of motile frozen-thawed testicular spermatozoa in secretory and excretory azoospermia. In excretory azoospermia, the number of motile spermatozoa was further increased when PTX was associated with migration on Percoll gradient, while PTX alone gave the best results in secretory azoospermia. Fertilization and pregnancy rates as well as embryo quality and division stages were comparable in the two groups.

By increasing the number of motile frozen-thawed testicular spermatozoa, PTX improves the selection of living spermatozoa.

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Correspondence to Nathalie Rives.

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Rives, N., Sibert, L., Mazurier, S. et al. Comment identifier le spermatozoïde vivant?. Androl. 12, 332–341 (2002). https://doi.org/10.1007/BF03034650

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