Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco School of Medicine, 550 16th Street, 7th Floor, San Francisco, CA, USA.
Stanford Canary Center for Early Cancer Detection, Stanford University, 3155 Porter Dr, Palo Alto, CA, USA.
Hum Reprod. 2018 Aug 1;33(8):1388-1393. doi: 10.1093/humrep/dey239.
Does microfluidic sorting improve the selection of sperm with lower DNA fragmentation over standard density-gradient centrifugation?
Microfluidic sorting of unprocessed semen allows for the selection of clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation.
Microfluidic devices have been explored to sort motile and morphologically normal sperm from a raw sample without centrifugation; however, it is uncertain whether DNA damage is reduced in this process.
STUDY DESIGN, SIZE, DURATION: This is a blinded, controlled laboratory study of differences in standard semen analysis parameters and the DNA fragmentation index (DFI) in split samples from infertile men (n = 70) that were discarded after routine semen analysis at an academic medical center.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm concentration, progressive motility and forward progression were assessed by microscopic examination. For each sample, the unprocessed semen was tested for DNA fragmentation and split for processing by density-gradient centrifugation with swim-up or sorting by a microfluidic chip. DNA fragmentation was assessed in unprocessed and processed samples by sperm chromatin dispersion assay. The DFI was calculated, from up to 300 cells per slide, as the number of cells with fragmented DNA divided by the number of cells counted per slide.
The median DFI in unprocessed samples was 21% (interquartile range (IQR): 14-30). In paired analyses of all samples, those processed by the microfluidic chip demonstrated significantly decreased DFI compared to those processed by density-gradient centrifugation (P = 0.0029) and unprocessed samples (P < 0.0001). The median DFI for chip specimens was 0% (IQR: 0-2.4) while those processed by density-gradient centrifugation had a median DFI of 6% (IQR: 2-11). Unprocessed samples in the highest DFI quartile (DFI range: 31-40%) had a median DFI of 15% (IQR: 11-19%) after density-gradient centrifugation and DFI of 0% (IQR: 0-1.9%) after processing with the microfluidic chip (P = 0.02).
LIMITATIONS, REASONS FOR CAUTION: While a high DFI has been associated with poor outcomes with IVF/ICSI, there are limited data illustrating improvements in clinical outcomes with a reduction in DFI. As this study utilized discarded, non-clinical samples, clinical outcomes data are not available.
While microfluidic sorting of unprocessed semen allowed for the selection of clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation, standard processing by density-gradient centrifugation with swim-up did not increase DNA fragmentation in an infertile population. The proposed microfluidic technology offers a flow-free approach to sort sperm, requiring no peripheral equipment or filtration step, while minimizing hands-on time.
STUDY FUNDING/COMPETING INTEREST(S): No external funding to declare. Utkan Demirci, PhD is the Co-founder and Scientific Advisor for DxNow Inc., LevitasBio Inc. and Koek Biotech. Mitchell Rosen, MD is a member of the Clinical Advisory Board for DxNow Inc.
微流控分选是否优于标准密度梯度离心法，改善精子 DNA 碎片率低的选择？
未处理精液的微流控分选可选择具有临床可用性、高活力且 DNA 碎片化程度几乎可检测到的精子。
已经探索了微流控装置来对原始样本中的运动和形态正常的精子进行分选，而无需离心；然而，在这个过程中是否降低了 DNA 损伤尚不确定。
研究设计、大小、持续时间：这是一项在学术医疗中心进行的盲法、对照实验室研究，比较了不育男性（n = 70）常规精液分析后丢弃的精液分析参数和 DNA 碎片指数（DFI）的差异。
参与者/材料、设置、方法：通过显微镜检查评估精子浓度、前向运动和前向进展。对于每个样本，用未处理的精液测试 DNA 碎片化，并通过密度梯度离心法的泳动或微流控芯片进行处理进行分割。用精子染色质弥散试验评估未处理和处理过的样本中的 DNA 碎片化。DFI 是通过每个载玻片最多 300 个细胞计算得出的，即具有碎片化 DNA 的细胞数除以每个载玻片上的细胞数。
未处理样本的中位数 DFI 为 21%（四分位距（IQR）：14-30）。在所有样本的配对分析中，与密度梯度离心法处理的样本（P = 0.0029）和未处理的样本（P < 0.0001）相比，用微流控芯片处理的样本 DFI 显著降低。芯片标本的中位数 DFI 为 0%（IQR：0-2.4），而密度梯度离心法处理的标本中位数 DFI 为 6%（IQR：2-11）。DFI 最高四分位数（DFI 范围：31-40%）的未处理样本经密度梯度离心后中位数 DFI 为 15%（IQR：11-19%），经微流控芯片处理后中位数 DFI 为 0%（IQR：0-1.9%）（P = 0.02）。
局限性、谨慎的原因：虽然高 DFI 与 IVF/ICSI 不良结局相关，但有数据表明降低 DFI 可改善临床结局。由于本研究使用了丢弃的非临床样本，因此无法获得临床结局数据。
虽然微流控分选未处理的精液可选择具有临床可用性、高活力且 DNA 碎片化程度几乎可检测到的精子，但标准的密度梯度离心法与泳动处理并没有增加不育人群的 DNA 碎片化。所提出的微流控技术提供了一种无流动的方法来分选精子，不需要外围设备或过滤步骤，同时最大限度地减少人工操作时间。
研究资金/竞争利益：没有外部资金申报。Utkan Demirci，博士是 DxNow Inc.、LevitasBio Inc. 和 Koek Biotech 的联合创始人兼科学顾问。Mitchell Rosen，医学博士是 DxNow Inc. 的临床顾问委员会成员。
