Résumé
A variety of natural and artificial cryoprotectant extenders have been explored to enhance sperm recovery following cryopreservation-thawing process.The current investigation is aimed at evaluating the effect of acetyl-L-carnitine on human spermatozoa and reactive species oxygen (ROS) level after freezing-thawing process.The spermatozoa were collected from 35 male patients diagnosed as having asthenospermia.The cryopreservation of human spermatozoa treated with acetyl-L-camitine at different concentrations (group B:2.5 mmol/L,group C:7.5 mmol/L,group D:15 mmol/L) was compared with control (group A:no acetyl-L-carnitine given).For the frozen-thawed spermatozoa,the viability,motility and DNA integrity were measured by comet assay,acrosome integrity by FITC-PNA staining and ROS level was determined in each group.The results showed that there were no significant differences in motility and viability between group A and group B,while the motility and viability of spermatozoa in group C and group D were significantly increased as compared with those in group A.As compared with group A,the values for DNA integrity parameters including comet rate (CR),tail DNA percentage (TD),tail length (TL) and Oliver tail moment (OTM) were significantly reduced in group C and group D.Group C and group D also displayed a higher proportion of intact acrosome than group A.No significant difference in ROS level was found between group A and group B,while with the increase in acetyl-L-camitine concentration,the ROS level in groups C and D was significantly reduced as compared with that in group A.In conclusion,acetyl-L-camitine at a concentration of 7.5 mmol/L is an effective antioxidant against cryo-damage on post-thawed human spermatozoa.
Résumé
La criopreservación del semen es una herramienta útil en la reproducción asistida, la cual puede tener impacto en las características espermáticas durante el congela miento y el descongelamiento. El objetivo de este estudio fue valorar la integridad del acroso ma y la movilidad de los espermatozoides criopreservados y descongelados provenientes de muestras hiperviscosas y no viscosas. Se realizó el espermograma, la integridad del acrosoma, el espermocultivo y los niveles de los marcadores de glándulas accesorias en 60 muestras de semen. Cada alícuota de semen fue inmersa en un crioprotector comercial para congelar a -196°C. Transcurridos 30 días, éstas fueron descongeladas y en el sedimento celular espermá ticesuspendido se evaluó la movilidad y la integridad acrosómica, disminuyendo significa tivamente la movilidad progresiva (p<0,05), la vitalidad espermática (p<0,005) y la integridad acrosómica (p<0,05); dicho descenso fue más evidente en las muestras hiperviscosas. La viscosidad del semen fresco se relacionó inversamente con la movilidad y la integridad del acrosoma antes y después del congelamiento (p<0,05). En veinte muestras de semen se iden tificó la presencia de microorganismos y de anticuerpos IgA anti C. trachomatis , de las cuales quince muestras en la reproducción hiperviscosas. El aumento de la viscosidad seminal y los niveles de ácido cítrico están asociados con disfunción prostática, baja movilidad espermática y reacción prematura del acrosoma, lo que puede reducir la capacidad fecundante de un esper matozoide. La etiología de la hiperviscosidad sigue siendo compleja; sin embargo, para pre servar la movilidad y la integridad del acrosoma, previamente deben investigarse sus causas en las muestras seminales que van a ser sometidas a la criopreservación.
Semen cryopreservation is a useful tool in assisted reproduction, which may have impact on sperm characteristics during freezing and thawing. The aim of this study was to assess the integrity of the acrosome and motility of cryopreserved and thawed spermatozoos in hyperviscous and no viscous samples. In semen samples spermiogram, glandular markers, acrosome integrity, culture and the levels markers accessory glands were measured. Each ali quot of semen was immersed in cryoprotectant and maintained in a commercial freezer at -196 ° C. After 30 days, these were thawed and in the cell pellet resuspended, spermatic motility and acrosomal integrity were evaluated. In thawed samples, there were significant decreases in progressive motility (p <0.05), vitality (p <0.005) and acrosome integrity (p <0.05) with respect to fresh sperm, this decline was most evident in hyperviscous samples. The viscosity of fresh semen was inversely related to motility and acrosome integrity before and after freezing (p <0.05). Twenty semen samples showed the presence of microorganisms and C. trachomatis IgA antibodies, of which fifteen showed hyperviscosity. Biochemical analysis demonstrated that semen samples with low levels of citric acid had less acrosomal integrity both before and after freezing (p <0.05). The viscoelasticity and citric acid levels are associated with prostate dys function, low sperm motility and premature acrosome reaction, which can reduce the fertilizing capacity of sperm. The etiology of hyperviscosity remains complex; however, to preserve mo tility and acrosome integrity, its causes must be investigated previously in the seminal samples to be subjected to cryopreservation.