密度梯度离心法在磁激活细胞分选前后：哪种技术对临床精子选择更有用？
Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection?
机构信息
Department of Reproduction and Development, Reproductive Biomedicine Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
出版信息
J Assist Reprod Genet. 2012 Jan;29(1):31-8. doi: 10.1007/s10815-011-9686-6. Epub 2011 Dec 15.
PURPOSE
Although, at present, the selection of sperm prior to ICSI is based on motility and morphology, undetectable anomalies, and more importantly damaged DNA are overlooked. In this regard, novel sperm selection procedures have gained much interest. For instance, sperm has been selected by Magnetic-Activated Cell Sorting (MACS) based on early apoptotic marker, the externalization of phosphatidylserine (EPS). Review of the literature has revealed that the efficiency of this technique has been mainly evaluated post Density Gradient Centrifugation (DGC). Therefore, there is a need to prove the efficiency of this technique independent of DGC. In addition, considering the fact that DGC induces EPS due to capacitation and acrosome reaction, therefore, the role of MACS before DGC(MACS-DGC) and MACS after DGC (DGC-MACS) should be assessed.
METHODS
Semen samples from fifteen infertile men were divided into three separate fractions: control, DGC, and MACS. To carry out DGC-MACS, DGC samples were further divided into two fractions and MACS was carried on the second fractions. Similarly to carry out MACS-DGC, the MACS samples were further divided into two fractions and DGC was carried on the second fractions. Percentages of sperm with normal morphology, DNA fragmentation, protamine deficiency, EPS and caspase-3 activity were determined in each fraction.
RESULTS
DGC is more efficient than MACS in separating intact sperm only in terms of normal morphology, DNA and chromatin integrity but not for active caspase. However, a combination of these procedures was more efficient than a single procedure to separate intact sperm for the aforementioned parameters. Comparison of the combined procedures showed only higher efficiency to separate active caspase in the MACS-DGC group.
CONCLUSION
Based on these results, we propose MACS-DGC rather than DGC-MACS to be implemented in clinical settings.
目的
尽管目前在 ICSI 之前选择精子是基于活力和形态，但无法检测到的异常，更重要的是受损的 DNA 被忽视了。在这方面，新的精子选择程序引起了广泛关注。例如，精子已经通过基于早期凋亡标记物（磷脂酰丝氨酸的外翻）的磁激活细胞分选（MACS）进行了选择。文献综述表明，这项技术的效率主要是在密度梯度离心（DGC）之后进行评估的。因此，需要证明这项技术在不依赖 DGC 的情况下的效率。此外，由于 DGC 诱导了顶体反应和 EPS，因此应该评估 MACS 在 DGC 之前（MACS-DGC）和 DGC 之后（DGC-MACS）的作用。
方法
将来自 15 名不育男性的精液样本分为三个单独的部分：对照组、DGC 和 MACS。为了进行 DGC-MACS，将 DGC 样本进一步分为两部分，并在第二部分进行 MACS。同样，为了进行 MACS-DGC，将 MACS 样本进一步分为两部分，并在第二部分进行 DGC。在每个部分中确定具有正常形态、DNA 碎片化、鱼精蛋白缺乏、EPS 和半胱天冬酶-3 活性的精子的百分比。
结果
DGC 在分离完整精子方面比 MACS 更有效，仅在正常形态、DNA 和染色质完整性方面，而不是在活性半胱天冬酶方面。然而，这些程序的组合在分离上述参数的完整精子方面比单一程序更有效。对联合程序的比较仅显示 MACS-DGC 组在分离活性半胱天冬酶方面具有更高的效率。
结论
基于这些结果，我们建议在临床环境中实施 MACS-DGC 而不是 DGC-MACS。
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