TITLE:
An Alternate Technique for Use of the ProteX/NovoSort Sperm Isolation System for Use with Low Volume-Low Concentration Cryopreserved Samples
AUTHORS:
Elliotte Cannon, Hannah Douglas, Lindsay Penrose, Samuel Prien
KEYWORDS:
Sperm Isolation, Cryopreserved, Non-Centrifuged, Barrier Isolation, Technique
JOURNAL NAME:
Advances in Reproductive Sciences,
Vol.13 No.3,
August
21,
2025
ABSTRACT: Centrifugation preparation has been the mainstay of ART sperm preparation since the procedure was first introduced in 1978. However, recent studies have suggested that commonly used collection and preparatory techniques, such as centrifugation, may negatively impact sperm cell function. Recently, a number of devices have been introduced to select sperm using a physical barrier. While these barrier techniques show great promise, most have been described as only working with freshly ejaculated sperm. As the less motile cryopreserved cells seem to lack the ability to penetrate the barriers. However, one of the newest systems, the NovoSort (NS; Reproductive Solutions Frisco, TX), uses a different barrier system that appears to harvest a higher percentage of motile cells and allows processing of whole ejaculates. The objective of the present study was to determine whether the system, with its unique barrier design, could be successfully adapted for use with frozen samples, particularly low-volume commercial samples, to enhance the motile population of sperm cells used in ART procedures. By design, the NS is intended for use with a ProteX collection cup (PX; Reproductive Solutions). The NS, containing 0.75 - 1.0 mL of media, is then lowered into the native ejaculate in the PX, allowing cells to migrate through the barrier into the clean media. While the design works well with a normal ejaculate, its design requires a volume of a minimum of 1 mL in the PX in order for it to make contact with the mesh of NS. As most commercial samples are both low volume (0.5 mL) and have relatively low motile concentrations, the standard technique is not an option. Therefore, the objective of the present study was to develop a technique that would allow the use of the NS barrier method with cryopreserved samples. In a series of preliminary experiments, it was found that the locations of the media and the native sample within the system could be reversed, creating the necessary contact between the sample, mesh, and media to facilitate sperm migration. To test the effectiveness of the modified protocol for motile cell recovery, 8 donated cryopreserved samples were thawed, and 0.5 mL of the sample, to mimic commercial samples, was placed in the NS. The NS was then placed in the PX, which contained 1 mL of culture media, and incubated for 1 hr. The outer media were then sampled at times 0, 5, 15, 30, 45, and 60 minutes to assess the presence and concentration of motile sperm. These data were then compared to the native concentration and forward progression at thaw. Results suggest these modifications allow for the recovery of a highly motile sperm population. There can be no doubt a significant amount of frozen semen is used in ART, especially in FDA-regulated male donor cases. Previous studies have suggested issues with motile cell recovery using other barrier methods due to decreased motility resulting from the freeze-thaw process. The current study suggests that, with modification, the combination of PX and NS can be employed to isolate motile cells from low–volume, low-concentration cryopreserved samples, such as commercial samples, thereby eliminating steps that might further damage compromised cells. Further study is needed to test the limits of this modified isolation technique.