Galmidi Bat-Sheva, Orvieto Raoul, Zurgil Naomi, Deutsch Mordechai, Fixler Dror
The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan 5290002, Israel.
Department of Gynecology and Fertility, Sheba Medical Center, Tel HaShomer, Ramat-Gan 5262000, Israel.
Int J Mol Sci. 2025 Jun 30;26(13):6343. doi: 10.3390/ijms26136343.
The cryopreservation of limited sperm samples, especially those retrieved from patients, poses significant challenges due to the small number of viable cells available for freezing. Traditional microliter cryopreservation methods are fraught with difficulties, as thawed sperm cells become nearly impossible to locate under a microscope due to their mobility and the multiple focal planes presented by larger drops. This search time is critical, as sperm cells enter a state of decline post thaw. Conversely, when sperm cells are cryopreserved in nanoliter volumes, they can be easily discovered but do not survive the freezing and thawing processes entirely. This phenomenon is attributed to the diffusion of water molecules from the droplet into the surrounding oil, which, while designed to limit evaporation, inadvertently increases solute concentrations in the aqueous environment, leading to cellular desiccation. This article elucidates the mechanisms underlying this lethal diffusion effect and presents a novel approach for freezing in nanoliter volumes, which has demonstrated significantly improved survival rates through carefully optimized procedures in clinical trials. Our findings highlight the importance of adapting cryopreservation techniques to enhance the viability of individual sperm cells, ultimately facilitating better outcomes in assisted reproductive technologies. This study provides the first quantification of nanoscale water diffusion dynamics during cryopreservation, establishing a predictive model that explains the catastrophic loss of sperm viability and identifying the critical role of water diffusion as a major impediment for limited samples. The novelty of our results lies in both elucidating this specific mechanism of cell death and introducing a novel approach: utilizing water-saturated oil as a protective layer. This method effectively mitigates the osmotic stress caused by water loss, demonstrating remarkably improved cell survival. This work not only advances the scientific understanding of cryopreservation at the nanoscale but also offers a practical, impactful solution poised to revolutionize fertility treatments for patients with low sperm counts and holds promise for broader applications in biological cryopreservation.
有限精子样本的冷冻保存，尤其是从患者体内获取的样本，由于可用于冷冻的活细胞数量少，面临着重大挑战。传统的微升冷冻保存方法充满困难，因为解冻后的精子细胞由于其流动性以及较大液滴呈现的多个焦平面，在显微镜下几乎无法定位。这个寻找时间至关重要，因为精子细胞解冻后会进入衰退状态。相反，当精子细胞以纳升体积进行冷冻保存时，它们很容易被发现，但并不能完全在冷冻和解冻过程中存活下来。这种现象归因于水分子从液滴扩散到周围的油中，虽然这旨在限制蒸发，但无意中增加了水环境中的溶质浓度，导致细胞脱水。本文阐明了这种致命扩散效应背后的机制，并提出了一种用于纳升体积冷冻的新方法，该方法在临床试验中通过精心优化的程序已证明存活率显著提高。我们的研究结果强调了调整冷冻保存技术以提高单个精子细胞活力的重要性，最终有助于辅助生殖技术取得更好的结果。这项研究首次对冷冻保存过程中的纳米级水扩散动力学进行了量化，建立了一个预测模型，该模型解释了精子活力的灾难性丧失，并确定了水扩散作为有限样本的主要障碍的关键作用。我们结果的新颖之处在于既阐明了这种特定的细胞死亡机制，又引入了一种新方法：利用水饱和油作为保护层。这种方法有效地减轻了因水分流失引起的渗透压力，显示出显著提高的细胞存活率。这项工作不仅推进了对纳米级冷冻保存的科学理解，还提供了一种实用、有影响力的解决方案，有望彻底改变精子数量低的患者的生育治疗方法，并有望在生物冷冻保存中得到更广泛的应用。
