IVF-Spain, Av. Ansaldo 13, 03540, Alicante, Spain.
Human Fertility Chair, University of Alicante, Carretera de San Vicente del Raspeig s/n, 03690, San Vicente del Raspeig, Alicante, Spain.
Hum Reprod. 2017 Oct 1;32(10):2007-2015. doi: 10.1093/humrep/dex281.
Is permeable cryoprotectant-free vitrification of native sperm samples a good alternative to conventional slow freezing?
The permeable cryoprotectant-free sperm vitrification protocol tested in this study renders considerably better recovery rates of good quality sperm compared to slow freezing.
Slow freezing is currently the most commonly used technique for sperm cryopreservation, though this method has been repeatedly shown to have negative effects on both structural and functional sperm features. New alternative methods such as vitrification have been established as a successful alternative in other reproductive cell types, but vitrification of spermatozoa is still a rather unexplored methodology, with limited studies showing its efficacy in male gametes.
STUDY DESIGN SIZE, DURATION: This study included 18 normozoospermic sperm samples from patients seeking ART treatment between 2014 and 2015. The effects of a new vitrification protocol on functional and structural sperm quality parameters in comparison to fresh and slow-frozen samples were assessed.
PARTICIPANTS/MATERIALS, SETTING, METHODS: All samples were divided into three aliquots: fresh (F), slow freezing-thawing (S) and vitrification-warming (V). Sperm concentration, motility, morphology, vitality, DNA fragmentation, cytoskeleton integrity and spontaneous acrosome reaction were assessed and compared between the groups.
Results showed improved preservation of sperm features after vitrification compared to conventional freezing. Permeable cryoprotectant-free vitrification presented a significantly higher percentage of live spermatozoa, than slow freezing, better preservation of acrosomes was achieved in vitrified samples and DNA fragmentation was reduced approximately one-third on average compared to slow freezing. Regarding tubulin assay, three different labelling patterns were observed. The frequency of these labelling patterns was similar in F and V groups but this was not the case of the S group. The multivariate analysis of all sperm quality parameters studied revealed that the V group presented features that are closer to the F group than the S group, indicating that samples are better preserved through vitrification than slow freezing.
This validation has been undertaken only on normozoospermic sperm samples. It would be necessary to compare these results in pathological samples and also to evaluate the influence of the application of this methodology on clinical outcomes.
The sperm vitrification protocol here described warrants better maintenance of sperm quality parameters than traditional freezing methods and may be a good alternative to preserve sperm samples from patients seeking IVF treatment.
STUDY FUNDING/COMPETING INTEREST(S): This study was funded by IVF-Spain Foundation. The authors have no conflicts of interest to declare.
原生精子样本的通透性无保护剂玻璃化冷冻是否是传统慢速冷冻的良好替代品？
与慢速冷冻相比，本研究中测试的通透性无保护剂精子玻璃化冷冻方案可显著提高优质精子的回收率。
慢速冷冻目前是最常用的精子冷冻保存技术，但这种方法已反复证明对精子的结构和功能特征都有负面影响。新的替代方法，如玻璃化，已被确立为其他生殖细胞类型的成功替代品，但精子的玻璃化仍然是一个相当未被探索的方法，只有有限的研究表明其在男性配子中的功效。
研究设计、大小、持续时间：本研究纳入了 2014 年至 2015 年期间寻求 ART 治疗的 18 名正常精子样本。评估了一种新的玻璃化方案对功能和结构精子质量参数的影响，与新鲜样本和慢速冷冻样本进行比较。
参与者/材料、设置、方法：所有样本均分为三份：新鲜（F）、慢速冷冻-解冻（S）和玻璃化-解冻（V）。评估并比较了各组的精子浓度、活力、形态、活力、DNA 碎片化、细胞骨架完整性和自发性顶体反应。
结果表明，与传统冷冻相比，玻璃化后精子特征的保存得到改善。与慢速冷冻相比，通透性无保护剂玻璃化可显著提高活精子的比例，玻璃化样本中顶体的保存更好，与慢速冷冻相比，DNA 碎片化平均减少约三分之一。关于微管蛋白分析，观察到三种不同的标记模式。F 和 V 组的这些标记模式的频率相似，但 S 组则不然。对所有研究的精子质量参数的多变量分析表明，V 组的特征与 F 组比 S 组更接近，这表明与慢速冷冻相比，玻璃化对样本的保存效果更好。
本验证仅在正常精子样本上进行。有必要在病理样本中比较这些结果，还需要评估该方法的应用对临床结果的影响。
这里描述的精子玻璃化方案比传统冷冻方法更好地维持精子质量参数，可能是保存寻求 IVF 治疗的患者精子样本的良好替代品。
研究资金/利益冲突：本研究由 IVF-Spain 基金会资助。作者没有利益冲突需要申报。
