P-986: Evaluation of seminal fractions using the Toluidine blue test and sperm chromatin structure assay

Abstract

Over the past decade, several techniques have emerged as potential protocols for evaluating the sperm chromatin integrity. Such integrity is of utmost importance for successful fertilization. Most of the techniques used for sperm chromatin evaluation require extensive instrumentation and special expertise. Toluidine blue (TB) test has been reported as an easily accessible alternative test for sperm chromatin assessment. Spermatozoa may either stain as dark violet (DV, abnormal chromatin conformation/DNA integrity) or light blue (LB, normal chromatin conformation). The sperm chromatin structure assay (SCSA) is the one of the most standardized methods for assessment of fragmented sperm DNA and the percentage of sperm with immature nuclear development (high DNA stainability index, %HDS). The objective of our study was to evaluate the Toluidine blue test and SCSA for assessment of sperm DNA damage in different seminal fraction. Prospective-controlled study. Semen samples were collected from 19 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated using the Toluidine blue test. In addition, the %HDS was measured using the SCSA. The means ± standard errors of the percentages of sperm with normal (light blue) and abnormal (dark violet) chromatin in neat semen, mature and immature fractions are given in the table below. Using the Toluidine blue test, mature sperm fractions showed higher incidence of spermatozoa with normal DNA compared to the immature fractions and neat semen samples (p=0.04 and p=0.006, respectively). Similarly, the mature sperm fractions showed lower incidence of spermatozoa with abnormal DNA compared to the immature fractions and neat semen samples (p=0.01 and p<0.0001, respectively). The %HDS showed a significant positive correlation with the number of DV sperm (r=0.51, p=0.0002) and a significant negative correlation with the number LB sperm (r=-0.59, p<0.0001).Tabled 1 Mature sperm fractions show a lower incidence of abnormal DNA as measured by the Toluidine blue test. The association of %HDS with cells that are stained dark violet suggests that Toluidine blue can identify immature nuclear chromatin with abnormal chromatin conformation.