Abstract
BackgroundThe use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. It has been hypothesized that the observed discrepancies could be attributed to the presence of apoptotic bodies that can be labeled with merocyanine 540, the so-called M540 bodies. In order to verify this hypothesis and determine the accuracy of our in-house FC-assisted evaluation of spermatozoa parameters, we used FC to evaluate both the fragmentation of sperm DNA using the TUNEL assay and the oxidation of sperm DNA using the 8-OHdG assay on semen samples with or without M540 bodies.ResultsWe show that the presence of M540 bodies lead to underestimation of both the level of sperm DNA fragmentation and sperm DNA oxidation when using FC assisted detection systems. We also observed that this situation is particularly pertinent in semen samples classified as abnormal with respect to the routine WHO semen evaluation as they appear to contain more M540 bodies than normal samples.ConclusionsWe conclude that M540 bodies interfere with both FC-conducted assays designed to evaluate sperm nuclear/DNA integrity. Exclusion of these contaminants in unprepared semen samples should be performed in order to correctly appreciate the true level of sperm DNA/nuclear damage which is known to be a critical male factor for reproductive success.
Highlights
The use of flow cytometry (FC) to evaluate sperm deoxyribo nucleic acid (DNA) fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses
In order to exclude the merocyanine 540 (M540) structures from the sample under analysis, we introduced a supplementary step to each FC protocol (SDF or sperm DNA oxidation (SDO)): propidium iodide (PI) was used following the rationale that unlike fixed dead spermatozoa, M540 bodies would not react with PI as they contain fragmented DNA in which PI could not intercalate [28, 29]
Despite the fact that non-cellular structures such as M540 bodies exist in semen and are present at a higher density in the semen of men with severely compromised spermatogenesis, clinicians and researchers studying sperm nuclear integrity by flow cytometry continue to ignore this issue
Summary
The use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. Over the past ten years, scientists and clinicians have agreed that condensation, fragmentation and, more recently, oxidation of the sperm nucleus are important parameters that determine the success of reproduction which go beyond fertilization success rate. This is relevant when considering the fidelity of embryonic development and the quality of life of the offspring. Even though it is not yet routinely examined, infertility clinicians have reached a consensus on the fact that sperm nuclear/DNA damage affects ART outcomes by challenging the oocyte repair capacity [2,3,4,5]. Sperm DNA/nuclear damage has far reaching consequences because if for any reason (e.g. extensive alterations to the sperm nucleus or/and a deficient oocyte repair system associated with advanced maternal age) paternal nuclear damage is not fully repaired by the oocyte, it can introduce de novo mutations that will impair embryo development and be transmitted to the generation potentially affecting offspring quality of life [5,6,7,8,9,10,11]
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