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Influence de la congélation sur le taux de fragmentation de l’ADN des spermes normaux à sévèrement altérés

Influence of cryopreservation on DNA fragmentation for normal to severely altered sperm

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La cryoconservation de spermatozoïdes est une technique couramment utilisée en AMP, en particulier dans les cas d’oligospermie sévère, dans la crainte d’une aggravation ultérieure. L’objectif de notre étude a été de rechercher un effet délétère éventuel du processus de migration-congélation-décongélation sur la fragmentation de l’ADN des spermatozoïdes évaluée par la technique TUNEL.

L’étude a porté sur 72 patients répartis en 4 groupes selon leurs caractéristiques spermatiques: groupe 1 [n=20] (paramètres «normaux» selon l’OMS), groupe 2 [n=24] (numération spermatique normale associée à une asthéno et/ou une tératospermie), groupe 3 [n=16] (numération totale comprise entre 5 et 20 millions), groupe 4 [n=12] (numération totale inférieure à 5 millions). Une évaluation des paramètres spermatiques conventionnels et de la fragmentation de l’ADN (technique TUNEL) a été réalisée sur le sperme entier (non migré non congelé) et sur le sperme décongelé. Une évaluation des taux de fragmentation a également été réalisée sur la fraction migrée avant congélation pour 20 patients (10 du groupe 1 et 10 du groupe 2); cette analyse n’a pas pu être effectuée sur les prélèvements oligospermiques (groupes 3 et 4).

Après l’ensemble du processus de migration, congélation et décongélation du sperme, le taux de fragmentation diminue dans la population témoin (groupe 1), alors qu’il augmente significativement dans les groupes de patients présentant des paramètres spermatiques altérés (groupes 2, 3 et 4). En examinant l’étape intermédiaire, la migration diminue les taux de fragmentation dans les 2 groupes étudiés par rapport au sperme entier.

Donc le taux de fragmentation diminue après migration, puis s’élève après congélation-décongélation à un taux inférieur à celui du sperme entier dans la population témoin, alors qu’il remonte à un taux plus élevé lorsque le sperme est altéré. Cependant, cet effet délétère de la congélation sur les spermes altérés reste modéré.

En conclusion, ces résultats sont plutôt en faveur de l’autoconservation de sperme pour les patients oligospermiques sévères. Correspondance:

Abstract

Ejaculated sperm cryopreservation can be proposed in the course of anART procedure, particularly in the case of severe oligozoospermia likely to deteriorate. The aim of this study was to evaluate the influence of the freezing-thawing process on sperm DNA fragmentation (analysed by the TUNEL technique).

The first step of this work consisted of adapting the TUNEL technique to perform this analysis on very poor quality sperm. A study was then performed on 72 patients divided into 4 groups according to their spermatic characteristics: group 1 [n=20] (“normal” parameters according to WHO), group 2 [n=24] (normal sperm count associated with asthenospermia and/or teratospermia), group 3 [n=16] (total sperm count between 5 and 20 M) and group 4 [n=12] (total sperm count below 5 M). Spermatic parameters and DNA fragmentation (performed by TUNEL in situ technique, 400 spermatozoa read per slide) were evaluated on raw semen - for all patients -, raw migrated sperm - for patients of group 1 and 2 -, migrated frozen-thawed sperm - for all patients-.

A TUNEL technique adapted to oligospermic samples was developed, manipulating spermatozoa directly on the slide rather than in suspension, to limit spermatic sample loss. After the whole migration-freezing-thawing process, the mean DNA fragmentation rate decreased for patients in group 1 (2.9 vs 5.1%, p<0.0001) whereas this rate increased for patients in groups 2 (10.5 vs 6.8%, p<0.0001), 3 (10.7 vs 7.6%, p<0.05) and 4 (15.2 vs 8.7%, p<0.005). DNA fragmentation rates from thawed samples were also correlated with initial spermatic parameters. At the intermediary step, migration decreased DNA fragmentation rate in comparison with raw semen rate in both groups (1.9 vs 4.7% [p<0.05] in group 1; 2.5 vs 5.4% [p<0.05] in group 2).

DNA fragmentation rate decreases after migration and then increases after freezing-thawing so that this rate is lower than the raw semen rate for “normal“ sperms and higher than the raw semen rate for altered sperms. Nevertheless, this DNA damage induced by cryopreservation on altered sperms remains moderate. Sperm “resistance” to cryopreservation also appears to depend on spermatic parameters. Cryopreservation may positively select spermatozoa, accelerating elimination of senescent spermatozoa by necrosis, so that early apoptotic spermatozoa from fresh ejaculate are not found in thawed samples.

These results, that need to be completed by a study on a larger sample of oligospermic patients, encourage us to continue cryopreserving severely altered sperms.

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Correspondence to Célia Sabbagh or Martine Albert or François Vialard or Ibrahim Hammoud or Marianne Bergere or Vincent Delabroye or Denise Molina-Gomes or Marc Bailly or Raoul Lombroso or Jacqueline Selval.

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Sabbagh, C., Albert, M., Vialard, F. et al. Influence de la congélation sur le taux de fragmentation de l’ADN des spermes normaux à sévèrement altérés. Androl. 17, 55–70 (2007) doi:10.1007/BF03041156

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Mots clés

  • cryoconservation de sperme
  • oligospermie sévère
  • fragmentation de l’ADN
  • TUNEL
  • migration spermatique
  • ICSI

Key-Words

  • sperm cryopreservation
  • severe oligozoospermia
  • DNA fragmentation
  • TUNEL
  • spermatic migration
  • ICSI