Comparison of three commercial sperm freezing media with regard to the influence on sperm motility, vitality and DNA fragmentation.

Background: Sperm cryopreservation is of fundamental importance for the assisted reproductive techniques. To date, there is still no uniform standard for sperm cryopreservation in the reproductive medical laboratories, although the quality of cryopreserved human sperm is strongly influenced by the choice of the freezing medium. Literature on the comparison of commercial sperm cryomedia still shows variable results depending on the study design, the tests conducted, and the different study populations investigated. The aim of our study was therefore to determine which of the three most commonly used commercial cryopreservation media, Sperm Freeze (SF; FertiPro), Sperm Freezing Medium (SFM; Cooper Surgical, Origio), and TEST Yolk Buffer (TYB; Fujifilm, Irvine Scientific), in combination with a slow-freezing protocol, is best suited for freezing ejaculates from infertile patients. Methods: Ejaculate leftovers (>1 mL) from 60 patients (age 16-57 years; mean 34.9 years) from the clinical diagnostic of the patients from the Andrological Department at the University Medical Center Hamburg-Eppendorf were collected over a period from November 2021 to May 2023. Each ejaculate rest was then divided into four portions of 250 µL, of which three were each frozen with one of three different commercial sperm cryomedia, SF, SFM and TYB. Frozen ejaculate portions were then thawed and analyzed with regard to the diagnostic sperm parameters motility, vitality and DNA integrity in comparison to the unfrozen (fresh) sample. To determine the motility of the sperm, a spermiogram was performed according to World Health Organization (WHO) criteria. Sperm vitality was examined by means of eosin staining. Sperm DNA integrity was investigated on the basis of a direct DNA fragmentation test [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay] and an indirect DNA fragmentation test [acridine orange (AO) staining]. DNA staining was analyzed applying the automated, microscope-based imaging platform ScanR (Olympus LS). Results: In the comparison of the three freezing media, TYB turned out to be the best regarding the sperm motility. In contrast, all three media performed equally in relation to the sperm vitality and preservation of sperm DNA integrity. Conclusions: Compared to SF and SFM, TYB can be recommended as the best medium for the cryopreservation of ejaculates from infertile patients. Nevertheless, since the ejaculates used for the analyses mostly came from infertile patients, results may not be representative for a "normal patient collective" and might therefore not be applicable, i.e., for sperm donors.