Cell-free DNA fragment size analysis in seminal plasma from men with azoospermia, oligozoospermia, and normozoospermia: a pilot study

Description

Introduction:
Approximately 50% of couple infertility cases are attributed to male factor, with one-third of these cases lacking clear aetiology. Cell-free DNA (cfDNA), circulating DNA found outside cells, offers various applications in medicine, including prenatal testing, cancer management, and therapy outcome prediction. Seminal plasma, comprising secretions from different seminal tract organs, may provide an information on function of these male reproductive organs. Some information exists on cfDNA concentrations in relation to sperm parameters, but no studies have analysed cfDNA fragment size profiles with a high sensitivity bioanalyzer. Therefore, our study aimed to analyse cfDNA fragment sizes in seminal plasma from men with 3 different phenotypes: azoospermia (AS), oligozoospermia (OS), and normozoospermia (NS).
Methods:
The study included 3 men with AS, another 3 men with OS, and 3 men with NS. Semen samples were collected after 2-7 days of sexual abstinence, centrifuged for cell removal, and stored at -270oC until cfDNA extraction. CfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit, and concentrations were measured before and after extraction with the Qubit™ dsDNA Quantification Assay Kit. Fragment length analysis was performed using the Agilent High Sensitivity DNA Kit.
Results:
CfDNA concentrations before extraction were comparable between AS, OS, and NS groups (3.06 ± 0.11 ng/µl, 3.75 ± 1.72 ng/µl, and 5.02 ± 2.57 ng/µl, respectively) (p>0.05). However, AS group show less intragroup variance. The NS group tended to have higher total ejaculate cfDNA concentrations (5.10 ± 1.55 µg in AS, 8.58 ± 2.98 µg in OS and 11.11 ± 4.59 µg in NS) (p>0.05).
CfDNA fragments ranged from ~50 bp to ~8000 bp, with no significant differences in average fragments frequency in size categories (<200 bp, 200-1000 bp, >1000 bp) between groups. Nevertheless, men in AS group are more similar in fragment distribution. On average, the most prevalent cfDNA fragments measure under 200 bp, representing short DNA fragments derived from mononucleosomal DNA at approximately 160-180 bp and ultra-short DNA fragments with more heterogeneous origins. Fragments over 1000 bp occur with similar or lower frequency than short DNA fragments and may originate from protamin-related DNA or genomic DNA in sperm. The least frequent fragments fall within the 200-1000 bp range.
While fragment profiles were generally similar, a patient with cryptozoospermia (OS group) exhibited a distinct profile with a high peak at 100 bp and fragments from 300 to 700 bp with about 30 bp periodicity.
Conclusions:
Our pilot study reveals that, on average, there is no significant difference in cfDNA concentration and fragment size profiles among men with azoospermia, oligozoospermia, and normozoospermia. This suggests that cfDNA concentration and fragment sizes in seminal plasma may not be directly influenced by the presence and concentrations of sperm cells in the ejaculate. This prompts questions about the origin of these fragments and their correlation with spermatogenesis. At the same time, individuals with azoospermia display more similarities in these measurements compared to other groups, warranting further analysis with an expanded participant pool.

Period	4 Sept 2024 → 8 Sept 2024
Event title	13th European Congress of Andrology (ECA2024)
Event type	Conference
Location	SwedenShow on map
Degree of Recognition	International