P-053 Comparative analysis between microfluidic sperm sorting and Swim-up technique to select spermatozoa with better characteristics in hyper viscous samples
Authors: Traini G , Tamburrino L , Ragosta M  E , Marcellini A , Vignozzi L , Baldi E , Marchiani S 
Published: July 2024

Abstract

Study question
What is the best technique to select spermatozoa in hyper viscous semen samples?

Summary answer
Microfluidics is the best technique that allows to select spermatozoa with better seminal characteristics and hyperactivated motility, and less sperm DNA fragmentation.

What is known already
Density gradient centrifugation and Swim-up (SU) are the two traditional selection methods used during assisted reproduction techniques (ART) to collect the highly motile sperm fraction. Many studies reported that centrifugation increase sperm DNA fragmentation and oxidative status which could affect reproductive outcomes. To overcome the limitations of centrifugation, an alternative approach by using microfluidic-based devices was recently designed. In microfluidic protocol, sample is applied to the device’s inlet port, connected to a lower chamber. This chamber consists of a microporous filter, where the most motile spermatozoa migrate upward through the filter.

Study design, size, duration
An observational study was conducted on 29 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from January 2023 to January 2024. In these samples we compare two different methods for sperm selection: indirect SU technique and microfluidic devices (MSS).

Participants/materials, setting, methods
We evaluated seminal parameters (sperm progressive and total motility, viability, and concentration), sperm kinematic parameters by Computer-Assisted Sperm Analysis (C.A.S.A. system), sperm DNA fragmentation by TUNEL/PI assay and sperm oxidative status by CellROX® Orange (which we have recently shown to identify a fraction of viable oxidized spermatozoa with better characteristics (Traini G., et al. 2022)) in 29 normozoospermic and 18 hyper viscous semen samples selected by SU and MSS.

Main results and the role of chance
In normozoospermic samples, sperm selection with both indirect SU technique and microfluidic devices, ameliorated sperm characteristics compared to not selected samples. Spermatozoa selected by MSS showed statistically significant higher progressive and total motility, viability and concentration compared to those selected by indirect SU. Moreover, an increase in most of kinematic semen parameters (like VAP, VCL and VSL) and hyperactivated motility was observed in MSS selected samples. Also, sperm DNA fragmentation was significantly reduced after MSS selection compared to indirect SU. We evaluated the percentage of viable oxidized spermatozoa by using CellROX® Orange. A significant increase of the percentage of CellROX® Orange positive spermatozoa was observed in most of MSS selected samples.
In all 18 hyper viscous samples, the results obtained in normozoospermic samples were confirmed. Indeed, an overall improvement of sperm progressive and total motility, viability and concentration together with an increase in most of kinematic semen parameters and in hyperactivated motility were evidenced in MSS selected spermatozoa respect to those selected by indirect SU. Again, a significant decrease of sperm DNA fragmentation and an increase of the percentage of CellROX® Orange positive spermatozoa, were observed after MSS selection.

Limitations, reasons for caution
The number of semen samples included in the study is not very elevated. Results obtained in hyper viscous semen samples should be confirmed in a greater number of samples.

Wider implications of the findings
Our data indicate that MSS selects the sperm fraction with better characteristics and less DNA fragmentation. These results suggest that MSS could improve ART outcomes, particularly for specific categories of patients, including hyper viscous semen samples, for which the selection by SU does not always guarantee good yields.

Trial registration number
not applicable