New insights in sperm biology: How benchside results in the search for molecular markers may help understand male infertility

Table 1 Promising sperm markers of male infertility based on so far published literature

Approach type	Main outcomes	Ref.	Advantages (+)/disadvantages (-)
Semen analysis
	Macroscopic and microscopic evaluation of semen according WHO guidelines	[2]	(+) Established reference values
			(-) High operator variability
			(-) Poorly predictive of fertility
Genetic and epigenetic
	NGS: Found a set of sperm RNA elements required to achieve live births	[47]	(+) Broad-spectrum analysis
	miRNA: Alteration of 5 miRNAs in subfertile and NOA subjects compared to controls	[46]	(-) Lack of validation
			(-) Not independently predictive of fertility
	DNA methylation: Different methylation pattern between fertile and infertile subjects	[42,43]	(-) Too early for diagnostic purpose
	sDF: Discrimination between fertile and infertile subjects	[55,56,58]	(+) Presently adopted in many ART laboratories
			(+) Prediction of fertility independent from semen quality
			(-) Employment of different techniques to detect sDF
			(-) Lack of agreement on cutoff values
Proteomic
	> 6000 proteins (histone variants, transcription factors, zinc finger proteins, receptors, proteins related to metabolism, structure and motility, carriers)	[80,95-98]	(+) Broad-spectrum analysis
			(-) Isolation of spermatozoa
			(-) Low available sperm material in oligozoospermic subjects
			(-) Intra- and inter-variability of proteomic profiles
PTMs
	Phosphorylation: Reduced tyrosine phosphorylation in asthenozoospermic subjects	[101]	(+) Higher biological relevance compared to gene or protein expression per se
	Ubiquitination: Sperm quality control system	[104]	(-) No target proteins identified
	Sumoylation: Marker of defective sperm	[107,108]	(-) Too early for diagnostic purpose
Ion channels
	Slo3: Involved in hyperpolarization during sperm capacitation	[111,112]	(+) Analysis free from confounders
	CatSper: Involved in sperm progressive and hyperactivated motility	[123]	(-) Skilled personnel and advanced instruments are required
			(-) Too early for diagnostic purpose

PTMs: Post-translational protein modifications; WHO: World Health Organization; NGS: Next-generation sequencing; NOA: Non-obstructive azoospermia; sDF: Sperm DNA fragmentation; ART: Assisted reproduction technique.