P–015 Characterization of ultrastructural morphology of human sperms by field-emission scanning electron microscopy using the NanoSuit method
Authors: So S , Takaku Y , Ohta I , Tawara F , Hariyama T 
Published: August 2021

Abstract

Study question
Can the NanoSuit method to observe sperm cells in wet conditions help treat male infertility using a field emission scanning electron microscope (FE-SEM)?

Summary answer
Compared with the conventional fixation method, the NanoSuit method can easily prepare FE-SEM samples without causing contraction and denaturation of human sperm cells.

What is known already
Evaluation of sperm morphology by optical microscopy is important for identifying male infertility. FE-SEM observation is useful for a more detailed evaluation of sperm morphology; however, a lot of the morphological information of the cells is lost by chemical fixation, dehydration, and freeze-drying. The NanoSuit method enables FE-SEM observation of unfixed cells under a high vacuum environment by electron beam polymerization of extracellular substances called NanoSuit. It has been reported that a sample prepared by the NanoSuit method retains the morphological information of live cells better than a sample prepared by the conventional fixation method.

Study design, size, duration
This laboratory study was conducted with informed consent and IRB approval. Semen parameters were within the WHO normal reference range.

Participants/materials, setting, methods
The conventional fixation method sample was prepared by fixing (glutaraldehyde and osmium), dehydration (ethanol and t-butyl alcohol), and freeze-drying. The NanoSuit method sample was introduced into the FE-SEM directly without conducting the above treatments. For observation, a JSM–7100F (JEOL, Japan) was used at an acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10–3 to 10–6 Pa.

Main results and the role of chance
Sperm head segmentation (acrosome, equatorial segment, and post acrosome), midpiece, and tail including endpiece could be clearly identified in the FE-SEM sample prepared by the NanoSuit method. Transmission electron microscopy revealed the existence of a thin polymerized extra layer, the NanoSuit, on the surface of the sperm. It is suggested that the presence of the NanoSuit layer enables FE-SEM observation of the unfixed sperm. The conventional fixation method causes a statistically significant contraction in the sperm head size compared to that calculated from optical micrographs (13.5 μm2 vs. 11.6 μm2, p &lt; 0.001). Furthermore, wheat germ agglutinin (WGA), a lectin, which is known to have the ability to bind to the sperm surface, did not bind to the fixed FE-SEM samples. This means that the original cell surface properties are lost in the fixed sperm sample. On the other hand, the FE-SEM sample prepared by the NanoSuit method did not show a statistically significant contraction of the sperm head compared to that calculated from optical micrographs (13.2 μm2 vs 12.9 μm2, p = 0.416); it also revealed a detailed binding pattern of gold-labelled WGA to the sperm surface. These results indicate that the NanoSuit method can prepare FE-SEM samples without sperm contraction and denaturation.

Limitations, reasons for caution
Characteristic sperm morphology in patients with male infertility should be investigated in future studies.
Wider implications of the findings: The NanoSuit method does not use chemical carcinogens and can prepare an FE-SEM sample in a shorter time than the conventional fixation method. The evaluation of ultrastructural morphology of unfixed sperms by this method may be useful for the identification of new morphological features and the evaluation of male infertility.

Trial registration number
Not applicable