Moving sperm forward

Abstract

Objective: The purpose of this study was to investigate a manual sperm sorting method (Swim-up) and a non-invasive microfluidics method (Zymot microfluidics) with emphasis on formation of reactive oxygen species (ROS) and DNA fragmentation as objective measurements of spermatozoa quality. Standard phase contrast imaging of spermatozoa was compared with quantitative phase contrast microscopy (QPM) imaging. Study design: Anonymized spermatozoa samples (n=9) from healthy men in North Norway were sorted with the Swim-up method and Zymot microfluidics (Zymot; Gaithersburg Maryland, USA). Analysis of ROS was done with an Oxisperm II kit (Halotech; Madrid, Spain) and by measuring MDA levels (Sigma-Aldrich; St. Louis USA) of spermatozoa. DNA fragmentation was visualized with spermatozoa chromatin staining using a Halosperm G2 kit (Halotech; Madrid, Spain) in conjunction with a Nikon ECLIPSE E 200 phase microscope (Nikon; Tokyo, Japan). QPM analysis was done with an in-house microscope and software. An un-paired T-test and ROUT test was completed to identify P-values and outliers. The software used for this was GraphPad Prism 9 version 9.0.0 (GraphPad; San Diego, California, USA)

Results: Native sperm and sperm plasma fractions had lower ROS levels in Swim-up samples compared to un-differentiated samples as analyzed with Oxisperm II, resulting in (P=0.0001). MDA-analysis showed that Swim-up and Zymot samples had lower ROS than Raw samples, showing a 0.05- and 0.7-fold difference respectively, resulting in P-value 0.0011 for Swim-up and P-value 0.0014 for Zymot. There was no significant difference in MDA levels between Swim-up and Zymot samples (P-value 0.6193). Swim-up and Zymot samples had a lower spermatozoa/ml than Raw samples, with the mean of Zymot and Swim-up being 460 and 877.7-fold lower. The resulting P-values were 0.001 for Zymot samples and <0.0001 for Swim-up samples. When comparing Swim-up and Zymot sample concentration, no difference was found as their datapoints were close to each other’s mean value (P-value 0.0829). DNA fragmentation was lower in Zymot and Swim-up samples compared with Raw samples, with P-values <0.0001 and 0.0047 respectively. Upon comparison, DNA fragmentation was found to be lower in Zymot samples than Swim-up samples with a P-value of 0.0230. The DNA fragmentation in Zymot spermatozoa was 1.2-fold lower than Swim-up. Outlier testing revealed 1 outlier in the Swim-up sample in the MDA analysis, using Q=1.000%. No other outliers were found. QPM provided live video of differentiated spermatozoa and created a heat map giving a “3D” topographical image. Regular phase microscopy was able to capture singular spermatozoa images stained with G2 DNA Fragmentation kit. QPM could not provide images of spermatozoa stained with the G2 DNA Fragmentation kit.

Conclusion: Levels of ROS were lower in Swim-up and Zymot sorted spermatozoa, indicating that these sorting methods can reduce ROS. MDA analysis showed no differences in ROS when comparing Swim-up and Zymot sorted spermatozoa. With regard to DNA fragmentation, it was demonstrated that Zymot isolated spermatozoa with less DNA fragmentation compared to Swim-up. QPM proved capable of live video data gathering of spermatozoa. However, quantitative analysis was hindered by lack of software tools. QPM was not able to capture high resolution images of DNA fragmentation due to the experimental setup being suboptimal.