P-077 The clues underneath the purple head: sperm fragmentation correlation with seminal quality parameters after swim-up techniques and sperm cryopreservation
Authors: Araujo Vieira B M , Ribeiro Â , Gomes A , Pinheiro de Magalhães R , Silva Carvalho J L 
Published: June 2023

Abstract

Study question
Does the culture media and the protocols used for sperm processing and cryopreservation significantly affect DNA integrity? How does it correlate to other sperm parameters?

Summary answer
In one of the analyzed media, the DNA damage was significantly higher after swim-up. DNA integrity is negatively correlated with concentration, vitality and motility.

What is known already
DNA fragmentation index (DFI) has proven to be a valuable tool in the diagnosis of infertility since men evaluated as normozoospermic in the seminogram may have high levels of sperm DNA fragmentation.There is already evidence of sperm DNA fragmentation affecting fertility, though the use of this information in ART is limited. The study of DNA fragmentation is relevant in fresh samples but also in processed and/or cryopreserved samples, since these procedures are likely to alter the sperm DNA integrity and change the treatment’s outcome. Extended culture times, media composition or even the performed protocol may induce those changes.

Study design, size, duration
The sperm sample collection was performed at CETI (Porto, Portugal) for 4 years. During this time, 207 seminogram samples were evaluated for DNA integrity using the Toluidine Blue test (TB) and Pearson’s correlation was applied. From that pool, 30 samples were selected to evaluate the impact of time and media used for swim-up protocols. Other 30 samples were analyzed for comparison after cryopreservation with two different media. In both cases an ANOVA test was applied.

Participants/materials, setting, methods
Men aged between 17 to 62 years old, collected a sperm sample for seminogram analysis according to the WHO and ESHRE criteria. After their informed consent was given, the same sample was used to test three different culture media. Swim-up technique was applied and the upper portion was removed for motility, concentration and DFI evaluation. Additionally, two distinct cryoprotectants (glycerol or egg yolk) were compared for sperm motility and DNA integrity before and after cryopreservation.

Main results and the role of chance
In this study, DNA integrity was evaluated using TB staining, a simple and affordable method for indirect assessment of DFI. DNA integrity was negatively correlated (p &lt; 0.0001) with concentration (r= -0.301), vitality (r= -0.421), morphology (r= -0.278) and total motility (r= -0.280). Moreover, normozoospermic samples showed higher DNA integrity than samples with at least one impaired parameter (p &lt; 0.0001). ROC curve analysis was used to establish a diagnostic threshold, above which samples are potentially damaged and consequently may lead to infertility situations. This value is around 21%, which could be an advance for the clinical application of this method, making the evaluation of DFI fair to all ART laboratories. Also, it was found that the incubation at room temperature may be beneficial since it improved sperm motility after 1h and 2h (respectively p = 0,0016 and p = 0,0130) compared to incubation at 37ºC with the same medium. All media recorded an increase in DNA damage over time. There were no significant statistical differences between cryopreservation media. However, the results suggest that the medium with egg yolk cryoprotectant may promote greater stability and membrane fluidity, decreasing the impact on sperm motility and DNA damage caused by the cryopreservation process.

Limitations, reasons for caution
These results should be taken with caution and, in order to strengthen them, additional samples should be analyzed. Also, the correlation between different sperm parameters is based on comparisons between several individuals who exhibit some interpersonal variability and was based on traditional semen analysis, which is slightly subjective.

Wider implications of the findings
The establishment of a threshold for DFI may be useful for daily practice. We observed that media rich in aminoacids and proteins promote sperm motility after swim-up and that room-temperature incubation is also beneficial. We concluded that media with egg yolk may promote greater sperm motility and less DNA damage.

Trial registration number
Not applicable