Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic

Abstract

The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for 30 s, followed by acridine orange staining. SCSA data from the male partners of 73 couples (group 1) achieving pregnancy during months 1-3 of Study I were used as the standard of 'sperm chromatin compatible with high fertility' and were significantly different from those of 40 couples (group 3) achieving pregnancy in months 4-12 (P < 0.01) and those of male partners of 31 couples (group 4) not achieving pregnancy (P < 0.001). Group 2 contained couples who had a miscarriage. SCSA values for Study II were almost twice that of the Study I fertility standards. Within-couple repeatability tended to be less for group 3 than for groups 1, 2 or 4. Based on logistic regression, spermatozoa with denatured DNA (cells outside the main population, COMP alpha t) were the best predictor for whether a couple would not achieve pregnancy. Some 84% of males in group 1 had COMP alpha t < 15%, while no couples achieved pregnancy in group 1 with > or = 30% COMP alpha t, a threshold level considered not compatible with good fertility. Using selected cut-off values for chromatin integrity, the SCSA data predicted seven of 18 miscarriages (39%).

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

“…Sperm DNA integrity was evaluated by SCSA as described elsewhere [12,21]. Briefly, fresh semen samples were incubated for 5 min in an ice-bath and an aliquot diluted to a sperm concentration of 1 × 10 6 -2 × 10 6 sperm per mL in TNE buffer (0.01 mol L -1 Tris-HCl, 0.15 mol L -1 NaCl, 1 mmol L -1 EDTA disodium, pH 7.4) in a total volume of 100 µL.…”

“…Recent reports have confirmed the utility of SCSA and concluded that the SCSA is the only method that can provide clear clinical threshold levels and can be recommended to provide robust evaluation of sperm DNA integrity [13,14]. Furthermore, it has been demonstrated that the SCSA can predict ART outcomes [4,12]. The etiology of sperm DNA damage appears to be multifactorial, including intrinsic factors such as DNA condensation failures and extrinsic factors such as air pollution, urogenital infections, fever, varicocele, certain prescription medications and chemotherapy, the exact mechanisms of which have not been elucidated [15].…”

“…The SCSA, introduced by Evenson et al [11], utilizes the metachromatic properties of the fluorescent stain, acridine orange (AO), and evaluates the extent of DNA denaturation at sites of acid-induced DNA strand breaks, which is determined by measuring the shift from green (double-stranded, native DNA) to red (single-stranded, denatured DNA) fluorescence by flow cytometry [11,12]. The SCSA parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), indicate the fraction of defective spermatozoa showing either the presence of DNA breaks or poorly condensed chromatin [11].…”

“…The SCSA parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), indicate the fraction of defective spermatozoa showing either the presence of DNA breaks or poorly condensed chromatin [11]. It has been shown that the chance of spontaneous pregnancy decreases when the DFI exceeds 20%-30% [12,13]. Recent reports have confirmed the utility of SCSA and concluded that the SCSA is the only method that can provide clear clinical threshold levels and can be recommended to provide robust evaluation of sperm DNA integrity [13,14].…”

Evaluating a novel panel of sperm function tests for utility in predicting intracytoplasmic sperm injection (ICSI) outcome

“…The need to reevaluate old test scores is best illustrated by how a reevaluation of the use of the SCSA DNA fragmentation index (DFI) scores has dramatically changed how the test results are used to direct patients to appropriate ART [16,17]. The SCSA is the only DNA integrity test with substantial scientific literature supporting the credibility of this test for use in diagnosing male infertility, and up until 2007, patients whose sperm had a DNA fragmentation index (DFI)≥30 were told that their sperm had minimal to no chance for successful ART attempts at pregnancy [22,31,32]. However, as the number of clinics using ICSI in treating their infertile couples, this DFI cut-off began to be questioned as many groups using sperm with DFIs of 30 or higher were having excellent live birth outcome rates [23,43,60].…”