Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa.

Alena J Hungerford, Hassan W Bakos, Robert John Aitken
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Abstract

Abstract: Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation.

Lay summary: Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

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冻存人精子分离方法分析。
摘要:精子冷冻保存是长期保存男性生育能力的重要手段。因此,确定解冻后分离精子的最佳技术对于确保以最小的医源性损伤恢复最高质量的精子至关重要。这不仅增加了成功受孕的机会,而且还降低了胚胎遗传损伤的风险。为了解决这个问题,人类精液样本使用慢速冷冻方案和Quinn的Advantage™精子冷冻培养基进行冷冻保存。随后将样品解冻并进行三种类型的精子分离程序:直接游泳、密度梯度离心和使用Felix™设备的电泳分离。低温保存导致预期的精子活力和活力的丧失,与脂质过氧化和DNA损伤的增加有关。在精子选择之后,所有三种分离技术都增加了精子活力,这在游泳和Felix™程序中尤为明显。后者还能显著提高精子活力。精子分离技术在形态方面没有差异,当考虑细胞活力时,线粒体活性氧的产生基本保持不变。相比之下,在DNA完整性和脂质醛形成方面观察到主要差异,其中Felix™分离的细胞表现出比其他分离程序明显更少的DNA损伤以及更低水平的4-羟基壬烯醛形成。因此,在分离配子的质量和实现分离所需的时间方面,电泳精子分离比其他分离策略具有显著的优势。概要:精子的长期储存对辅助生殖技术至关重要,因为它可以保存可能由于化疗或输精管切除术等因素而受到损害的生育能力。这一目标可以通过冷冻保存来实现——将细胞冷冻到-196°C。当精子随后需要受孕时,必须小心地将它们从冷冻保存介质中分离出来,以最大限度地提高成功受孕的机会,并最大限度地降低后代遗传缺陷的风险。本文比较了三种分离技术从冷冻保存后的精液和培养基中分离理想精子的能力。研究发现,低温保存会降低细胞的运动性和活力,并造成更高水平的DNA和细胞膜损伤。在比较的三种技术中,只有根据其大小和电荷(电泳分离)分离的细胞表现出明显较低的DNA片段化水平。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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