Maleki Behnam, Khalili Mohammad Ali, Gholizadeh Lida, Mangoli Esmat, Agha-Rahimi Azam
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Cryobiology. 2022 Feb;104:15-22. doi: 10.1016/j.cryobiol.2021.11.176. Epub 2021 Nov 22.
Testicular sperm extraction (TESE) is an invasive surgery for achieving the spermatozoa in cases with azoospermia. In these patients, the number of retrieved spermatozoa is limited and the optimal cryo-storage is very critical for their fertility preservation. Therefore, single sperm vitrification has been introduced for preservation of low number of spermatozoa. The goal was to assess the efficacy of sperm freezing medium (SFM) and sucrose medium as cryoprotectants for single sperm vitrification in cases with severe oligozoospermia and azoospermia. A total of 20 ejaculates from severe oligozoospermia and 20 testicular samples from azoospermia were processed. Twenty-five sperm cells were collected using ICSI injection pipette and transferred to a cryoprotectant droplet placed on the Cryotech, then vitrified by plunging in liquid nitrogen. Sperm motility, viability, fine-morphology, mitochondrial activity and DNA fragmentation index (DFI) were assessed before and after vitrification. Sperm motility, viability and the percentage of cells with mitochondrial activity were significantly decreased after vitrification in both severe oligozoospermic and testicular samples in either cryoprotectants. However, the rates of post-warm sperm motility and the cells with mitochondrial activity increased significantly in sucrose medium in both severe oligozoospermic and testicular samples compared to SFM. In testicular samples, the DFI of spermatozoa vitrified in SFM was significantly higher than those vitrified with sucrose medium. Sperm motility, viability, mitochondrial activity, and DNA integrity were better preserved in sucrose medium than SFM after single cell vitrification. The presented method may be a useful candidate for successful freezing of individual sperm cells in clinical setting.
睾丸精子提取术（TESE）是一种用于在无精子症病例中获取精子的侵入性手术。在这些患者中，获取的精子数量有限，而最佳的冷冻保存对于他们的生育力保存至关重要。因此，已引入单精子玻璃化冷冻技术来保存少量精子。目的是评估精子冷冻培养基（SFM）和蔗糖培养基作为冷冻保护剂在严重少精子症和无精子症病例中单精子玻璃化冷冻中的效果。对来自严重少精子症的20份精液和来自无精子症的20份睾丸样本进行了处理。使用卵胞浆内单精子注射（ICSI）注射移液器收集25个精子细胞，并将其转移到置于Cryotech上的冷冻保护剂液滴中，然后投入液氮中进行玻璃化冷冻。在玻璃化冷冻前后评估精子活力、存活率、精细形态、线粒体活性和DNA碎片指数（DFI）。在两种冷冻保护剂中，严重少精子症和睾丸样本在玻璃化冷冻后精子活力、存活率和具有线粒体活性的细胞百分比均显著降低。然而，与SFM相比，在严重少精子症和睾丸样本中，蔗糖培养基中复温后的精子活力和具有线粒体活性的细胞率均显著增加。在睾丸样本中，SFM中玻璃化冷冻的精子的DFI显著高于蔗糖培养基中玻璃化冷冻的精子。单精子玻璃化冷冻后，蔗糖培养基比SFM能更好地保存精子活力、存活率、线粒体活性和DNA完整性。所提出的方法可能是临床环境中成功冷冻单个精子细胞的一种有用方法。
