Membrane-bound receptor for advanced glycation end products (RAGE) is a stable biomarker of low-quality sperm
Authors: Browning Jill , Ghanim Magda , Jagoe William , Cullinane Jennifer , Glover Louise E , Wingfield Mary , Kelly Vincent P 
Published: November 2024

Abstract

STUDY QUESTION
Does receptor for advanced glycation end products (RAGE) on the surface membrane of the sperm cell function as a biomarker of low-quality sperm?

SUMMARY ANSWER
Membrane-bound RAGE at a cellular level directly correlates with low sperm motility, high cell permeability, decreased mitochondrial function, DNA fragmentation, and higher levels of apoptosis.

WHAT IS KNOWN ALREADY
RAGE has previously been measured by ELISA in low-quality sperm in diabetic men and has been shown to correlate with DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay).

STUDY DESIGN, SIZE, DURATION
Semen samples were recovered from 60 non-obese, non-diabetic and non-smoking subjects, washed with fresh media, and analysed directly or purified further by differential gradient centrifugation (DGC) or fractionated by direct swim-up before being analysed for sperm motility and molecular health parameters, including cell membrane permeability, cell death, mitochondrial membrane potential, DNA fragmentation, and RAGE protein expression.

PARTICIPANTS/MATERIALS, SETTING, METHODS
Sperm motility assessments were carried out by computer-assisted sperm analysis (CASA) on 1000 spermatozoa for washed samples and 300 spermatozoa for purified samples. Molecular sperm health parameters were evaluated using flow cytometry with the use of the following markers: DAPI for cell membrane permeability, Annexin V/DAPI for cell death (apoptosis and necrosis), MitoTracker® Red CMXRos for mitochondrial membrane potential, TUNEL assay for DNA fragmentation and 8-hydroxy-2-deoxyguanosine for identification of oxidative damage to sperm DNA, and contrasted to membrane-bound RAGE expression levels, which were evaluated using an anti-RAGE monoclonal mouse antibody.

MAIN RESULTS AND THE ROLE OF CHANCE
RAGE protein was shown to be present on the acrosomal and equatorial regions of sperm, with the levels of membrane bound receptor strongly correlating with poor sperm health across all parameters tested; motility (R2 = 0.5441, P &lt; 0.0001) and mitochondrial membrane potential (R2 = 0.6181, P &lt; 0.0001) being of particular note. The analysis was performed at a single cell level thereby removing confounding complications from soluble forms of the RAGE protein that can be found in seminal plasma. The expression of the RAGE protein was shown to be stable over time and its levels are therefore not subject to variation in sample handling or preparation time.

LARGE SCALE DATA
N/A.

LIMITATIONS, REASONS FOR CAUTION
Inclusion criteria for this study were non-diabetic, non-obese and non-smoking participants to assess the distribution of RAGE expression in the general population, thereby excluding disease conditions that may increase RAGE expression in sperm or contribute to low sperm quality. The study does not address how RAGE expression may be affected in other patient subpopulations or disease states associated with male infertility. Sperm analysis by flow cytometry is not amenable to the study of males with a low sperm count.

WIDER IMPLICATIONS OF THE FINDINGS
Results of this study suggest that RAGE expression is a molecular maker of sperm cell health, which may be used for improvements in assisted reproduction through the removal of RAGE expressing sperm and facilitate in the diagnoses of unexplained infertility through its use as a biomarker of male infertility.

STUDY FUNDING/COMPETING INTEREST(S)
The study was funded by the Irish Research Council under the Government of Ireland Programme (GOIPG/2015/3729) and the Enterprise Ireland Innovation Partnership Programme (IP-2020-0952). All authors declare no competing interests.