A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index

Abstract

SummaryThe impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

Evaluation of sperm chromatin structure and DNA strand breaks is an important part of clinical male fertility assessment

“…The final structure of sperm chromatin is dependent upon post-translational methylation and acetylation that affects chromatin stability and the acquisition of epigenetic/imprinting marks impacting on embryonic development. This faulty compaction makes an abnormal tertiary chromatin structure that is crucial for correct timing during the process of fertilization and early pre-implantation development ( 21 , 22 ). The threshold for HDS related embryo failure is 20% to >25% HDS; 22% of 1,417 infertility patients were at these levels ( 23 ).…”

“…Figure 2 graphically summarizes the three thresholds for SCSA: (I) <20%; (II) >25%; (III) 30–40%. The 20–25% DFI has been considered the “grey zone” by Spano et al ( 27 ), Erenpreiss et al ( 31 ), Oleszczuk et al ( 30 ) and Hamadi et al ( 21 ) and fertility problems may start to occur when SCSA DFI reaches this level. It is noted, however, that our SDI clinical service has seen natural full-term pregnancy with up to 68% DFI.…”

Evaluation of sperm DNA structure, fragmentation and decondensation: an essential tool in the assessment of male infertility

“…Acridine orange/flow cytometry is routinely used in our practice, using a protocol as previously described ( 14 ). In order to monitor the reliability of the method, samples are randomly re-tested every week (internal standards).…”

Abnormal Chromatin Condensation in Spermatozoa and Dna Fragmentation in Spermatozoa: Is There a Correlation?

“…В последнее десятилетие опубликованы многочи-сленные исследования, авторы которых пытаются уста-новить корреляцию между фрагментацией ДНК и ста-тусом хроматина [30,[42][43][44][45][46].…”

“…В связи с этим возник интерес к вопросу корреляции этих типов атипии. Многочисленные исследования, в которых результат ремоделирования хроматина опре-деляли окраской хромомицином А3 либо анилиновым синим, а ФДС -с помощью COMET, SCSA или TUNEL, демонстрировали наличие корреляции между этими процессами [42,43,45]. В отличие от соматических клеток, в сперматозоидах ФДС не является показателем апоптоза [107].…”