Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.

Cytoskeleton Structure in Mouse Sperm and Testes After 30 Days of Hindlimb Unloading and 12 Hours of Recovery.

BACKGROUND/AIMS: Changes in the external mechanical field result in cytoskeleton reorganization and the formation of adaptive patterns in different types of cells, including somatic cells and sex cells. The aim of this research was to study the protein and mRNA content of cytoskeletal and sperm-specific genes in the sperm and testis cells of mice. METHODS: Mice were subjected to 30 days of antiorthostatic suspension to simulate weightlessness, followed by 12 h of recovery, while receiving essential phospholipids at a dosage of 500 mg/kg/day (30HSE and 30HSE+12h groups) or a similar dosage of a placebo (30HS and 30HS+12h groups). Accordingly, reference groups (CE group and C group) were formed. The total number and the percentage of motile spermatozoa were calculated using a Makler chamber. To analyze the number of viable spermatozoa and the permeability of their membranes, eosin staining was used as well as Diff-Quick for a morphological evaluation. Relative protein and mRNA content was estimated in a western blot and quantitative PCR assay, respectively. RESULTS: The relative protein expression levels of actin (beta and gamma) and two alpha-actinin isoforms (1 and 4) remained constant in the sperm of all study groups, except for the 30HS+12h group, where the alpha-actinin-4 level was 13% higher than in the reference group (p < 0.1). In the testis cells, the relative actin isoform content was equivalent to that in the spermatozoa. However, in the testis cells, the ACTN1 mRNA content was 17% higher in the 30HS group than in the C group (p < 0.05), and decreased after 12 h of recovery. In contrast, the ACTN4 mRNA content was 20% lower in the 30HS group than in the reference group (p < 0.05) and increased after the 12-h recovery period. At the same time, in the group administered the essential phospholipids, the relative ACTN1 and ACTN4 mRNA content did not differ from those of the reference group. The relative beta-tubulin content was similar in the reference C group and the reference CE group, which was administered the essential phospholipids. In the 30HS and 30HS+12h groups, the beta-tubulin content decreased by 19% and 22% (p < 0.05), respectively, and they also decreased in the groups administered the essential phospholipids (30HSE and 30HSE+12h groups, by 27% and 33%, respectively, p < 0.05). In the testis tissue, the relative tubulin content did not change in any of the experimental groups. At the same time, the relative mRNA content of the genes encoding the studied cytoskeletal proteins increased, which may indicate the protein content was regulated mainly at the translational level. CONCLUSION: The spermogram parameters and the content of the sperm-specific proteins and the associated mRNAs revealed a decrease in the number of mature spermatozoa in mice suspended under conditions of weightlessness. Moreover, the decrease was prevented by the administration of essential phospholipids.