• 作者：Leonor Ortega Ló ; pez ; ^{lortega@clinicatambre.com} ; Enrique Olaya Vila ; Patricia Ló ; pez Domí ; nguez ; Aurora Gabriel Segovia ; Ivá ; n Orozco Gó ; mez ; Rocí ; o Nú ; ñ ; ez Calonge ; Pedro Caballero Peregrí ; n
• 关键词：Fragmentació ; n ADN ; Vitalidad ; Naranja de acridinabromuro de etidio ; SCD
• 刊名：Revista Internacional de Androlog铆a
• 出版年：2010
• 期刊代码：pca322_1698031X
• 类别：med
• 出版时间：July-September 2010
• 卷：8
• 期：3
• 页码：114-121
• 文件大小：158 K

Introduction

Sperm DNA integrity is one of the most important factors currently analyzed in the study of male fertility. There are numerous publications on the utility of diverse methods analyzing sperm chromatin status. One of the simplest is the ethidium bromide/acridine orange (AO) test, which assesses the degree of sperm DNA denaturation according to its binding to acridine orange staining. However, the AO test is limited to these metachromatic properties and consequently provides limited information on the nuclear status of the sperm. Other techniques, such as the sperm chromatin dispersion (SCD) test, analyze sperm DNA fragmentation by assessing DNA strand breaks.

Objective

To analyze whether there is a correlation between sperm vitality measured through the ethidium bromide/AO test and DNA fragmentation measured through the SCD test. In both cases, the samples were analyzed visually.

Material and methods

Eighty-two semen samples from men undergoing fertility testing in the Cl铆nica Tambre were included in this study. The ethidium bromide/AO test was used to test sperm vitality and the SCD test (Halosperm, Halotech) was used to evaluate sperm DNA fragmentation. The AO technique detects the proportion of dead sperm, depending on the degree nuclear DNA denaturation. Sperm with orange fluorescence indicate denatured DNA, while those with green fluorescence contain intact DNA.

The percentage of fragmentation was measured by the improved SCD test (Halosperm). This test indicates DNA strand breaks based on the size of the halo obtained. In fragmented sperm, no halo is observed when the sample is examined with a fluorescence microscope and GelRed fluorochrome. Sperm with intact DNA show a halo.

Student's t-test was used to compare the means between the two groups. Differences of p<0.05 were considered statistically significant.

Results

The proportion of sperm with denatured DNA detected with the AO test was significantly correlated with the proportion of sperm DNA fragmentation detected with the SCD test (r = 0.5, P < 0.001). In the 82 samples analyzed, the mean vitality index with the AO test was 20.93%(6 卤 50) versus 28.35%(7.3 卤 40) for the fragmentation index. The proportion of sperm with denatured or fragmented DNA was also positively correlated with motility. The two assays showed a statistically significant negative correlation with sperm concentration.

Discussion

This study demonstrates a correlation between the results of sperm vitality measured by the AO test and the degree of DNA fragmentation measured by the SCD test. These results suggest that the AO test could be the first option to determine sperm chromatin damage before more expensive or complicated tests are performed.