Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential
Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential
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DOI：
10.1186/1477-7827-7-1
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发表时间：
2009-01-08
影响因子：
4.4
通讯作者：
de Agostini, Ariane
中科院分区：
文献类型：
--
作者：
Arrighi, Corinne de Vantery;Lucas, Herve;de Agostini, Ariane
背景资料：磷脂酰丝氨酸（EPS）的外化发生在类精子中，并可用于将其从精子制备物中去除，以提高精子质量，用于辅助医疗生殖。我们首先根据精子EPS的频率以及精子上磷脂酰丝氨酸（PS）的定位来表征不育患者精子中的EPS。随后，我们确定了耗尽EPS精子对精子quality.Methods的影响：EPS可视化荧光标记膜联蛋白V结合试验。Annexin V和Hoechst双重染色可区分凋亡精子和坏死精子。我们使用磁激活细胞分选膜联蛋白V结合微珠（MACS-ANMB）技术，以去除密度梯度离心（DGC）制备的精子EPS精子。采用罗丹明123测定精子的前向运动能力、活力和线粒体膜电位（MMP）的完整性，评价该技术对精子质量的影响。结果：DGC精子中EPS精子的平均百分比为14%。四个亚群的精子被确定为：70%的活，3%的早期凋亡，16%的坏死和11%的晚期凋亡或坏死。PS主要分布在精子头部和/或中段或整个精子上。MACS可有效清除EPS精子。MACS结合DGC允许平均减少70%的EPS和60%的MMP-破坏的精子，平均增加50%的精子存活率在24小时。结论：人类精液中含有EPS精子，大多可以消除DGC加MACS，从而提高精子的长期生存能力，活力和MMP的完整性。EPS可用作精子质量的指标，去除EPS精子可提高辅助医疗生殖的生育能力。
Background: Externalization of phosphatidylserine (EPS) occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS) on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality.Methods: EPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB) technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC). The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP) by Rhodamine 123.Results: Mean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h.Conclusion: Human ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation.