Infertility is a widespread problem, and in some cases, the routine basic semen analysis is not sufficient to detect the cause of male infertility. The use of the scanning electron microscope (SEM) could provide a detailed insight into spermatozoa morphology, but it requires specific sample preparation techniques. The purpose of this study was to select, adjust, and optimize a method for the preparation of spermatozoa samples prior to SEM analysis, and to establish the protocol required for its use in clinical practice. We examined sperm samples of 50 men. The samples were fixed with modified iso-osmolar aldehyde solution followed by osmium post-fixation. In the first method, dehydration of the cells and subsequent critical point drying (CPD) were performed on a coverslip. In the second method, the samples were dehydrated in centrifuge tubes; hexamethyldisilazane (HMDS) was used as a drying agent instead of CPD, and the samples were air-dried. The third procedure was based on a membrane filter. The samples were dehydrated and dried with HMDS in a Gooch crucible, continuously, without centrifugation or redispersion of the sample. Our results showed that the fixation with modified iso-osmolar aldehyde solution followed by osmium post-fixation, and combined with dehydration and CPD on a coverslip, is the most convenient procedure for SEM sample preparation. In the case of small-size samples or low sperm concentration, dehydration and drying with HMDS on the membrane filter enabled the best reliability, repeatability, and comparability of the results. The presented procedures are suitable for routine use, and they can be applied to confirm as well as to correct a diagnosis.

中文翻译：

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采用扫描电子显微镜 (SEM) 评估人类精子形态的方法。

不育是一个普遍存在的问题，在某些情况下，常规的基本精液分析不足以检测男性不育的原因。使用扫描电子显微镜 (SEM) 可以详细了解精子形态，但需要特定的样品制备技术。本研究的目的是选择、调整和优化扫描电镜分析前精子样品的制备方法，并建立其在临床实践中使用所需的方案。我们检查了 50 名男性的精子样本。样品用改良的等渗醛溶液固定，然后用锇进行后固定。在第一种方法中，细胞脱水和随后的临界点干燥（CPD）在盖玻片上进行。第二种方法，样品在离心管中脱水；使用六甲基二硅氮烷（HMDS）代替CPD作为干燥剂，并将样品风干。第三个程序基于膜过滤器。样品在古氏坩埚中用 HMDS 连续脱水和干燥，无需离心或再分散样品。我们的结果表明，用改良的等渗醛溶液固定，然后进行锇后固定，并结合脱水和盖玻片上的 CPD，是 SEM 样品制备最方便的程序。对于小尺寸样品或精子浓度较低的情况，在膜过滤器上使用 HMDS 脱水和干燥可实现结果的最佳可靠性、重复性和可比性。所提出的程序适合常规使用，并且可用于确认和纠正诊断。