Sadeghi Niloofar, Tavalaee Marziyeh, Kiani-Esfahani Abbas, Moazamian Aron, Gharagozloo Parviz, Aitken Robert J, Drevet Joël R, Nasr-Esfahani Mohammad Hossein
Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
CelloXess LLC, 830 Bear Tavern Road, Ewing, NJ, 08628, USA.
Basic Clin Androl. 2021 Oct 21;31(1):23. doi: 10.1186/s12610-021-00143-7.
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. 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.
We 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.
We 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.
与传统荧光显微镜分析相比，利用流式细胞术（FC）通过脱氧核苷酸末端转移酶介导的缺口末端荧光素dUTP标记法（TUNEL）评估精子DNA片段化情况时结果并不一致。据推测，观察到的差异可能归因于凋亡小体的存在，这些凋亡小体可被部花青540标记，即所谓的M540小体。为了验证这一假设并确定我们内部使用FC辅助评估精子参数的准确性，我们使用FC对含有或不含有M540小体的精液样本进行TUNEL检测评估精子DNA片段化情况，并使用8-羟基脱氧鸟苷（8-OHdG）检测评估精子DNA氧化情况。
我们发现，使用FC辅助检测系统时，M540小体的存在会导致对精子DNA片段化水平和精子DNA氧化水平的低估。我们还观察到，这种情况在世界卫生组织（WHO）常规精液评估分类为异常的精液样本中尤为明显，因为这些样本似乎比正常样本含有更多的M540小体。
我们得出结论，M540小体干扰了旨在评估精子核/DNA完整性的两种FC检测方法。为了正确评估精子DNA/核损伤的真实水平，已知这是生殖成功的关键男性因素，应在未处理的精液样本中排除这些污染物。
