Authors: Broussard A^1,2,3, Leader B², Tirado E², Colver R¹, Reuter L¹, Bopp B¹, Will M¹, Anspach Will E¹, Adaniya G⁴, ¹Midwest Fertility Specialists, ²ReproSource, ³Eastern Virginia Medical School, ⁴Ovation Fertility Indianapolis

Objective: This was a prospective study to compare different commercially available sperm preparation techniques on the same semen sample to determine which methods most effectively improves sperm DNA fragmentation index (DFI) and other sperm health biomarkers, such as oxidative stress adducts (OSA) and high DNA stainability (HDS) index.

Design: Sperm DNA fragmentation (SDF) has previously been correlated with adverse outcomes in the in vitro fertilization (IVF) laboratory, poor implantation and increased pregnancy loss in multiple studies. In a previous study we determined that sperm DNA fragmentation index (DFI), oxidative stress adducts (OSA), and high DNA stainability (HDS)   at the time of insemination was negatively correlated with fertilization rates, with both standard insemination and intracytoplasmic sperm injection (ICSI). We concluded that different sperm preparation methods should be evaluated to determine which yielded the best sperm quality.

In this current study we evaluated different methods to improve DFI, OSA, and HDS. The methods used were ZyMōt Sperm Separation Device (1,DxNow) Isolate gradient (2), SpermGrad gradient (3), Isolate + Swim-up(4), and SpermGrad + Swim-up (5). Semen samples of 30 men with the  mean age of 37±8.3 years were processed with each method. Neat sperm concentrations ranged from 7 to 104 million/mL with 23.3% oligospermic. The motility of the samples ranged from 15% to 80% motility with an average of 52.2±16.9%. The sperm separation device consistently outperformed the other methods to improve DFI, HDS, and OSA.

Materials and Methods: Coded specimens were sent to an external clinical laboratory (ReproSource, Woburn, MA) for blinded SDF measurement, reported as % DNA Fragmentation Index (%DFI), using the acridine orange and flow cytometry as described in the SCSA^© method. DFI, OSA, and HDS were compared between preparation methods on the same ejaculate using Wilcoxon Rank Sums between each pair with P<0.05 considered significant (See table). Box and whisker plots were used to display the variation of DFI, HDS, and OSA post-processing.

Results:

Conclusions: The sperm separation device is a novel method that effectively reduced DFI (p<0.0001) compared to two different gradients (2,3) and gradients followed by swim-up (4,5). The device also has eliminated centrifugation, a step known to increase oxidative stress. The device reduced OSA levels, a measurement of oxidative stress, and HDS, which is a measurement of immature cells and high histone retention. These are both indicators of sperm health and function. Overall, the quality of the sperm obtained post-processing was improved by the use of the separation device, which may increase the chance of a healthy sperm being used for fertilization.

Support: This study was possible by the support of a ReproSource Investigator Reward and the donation of the ZyMōt devices from DxNow.

Poster