Apoptotic sperm biomarkers and their correlation with conventional sperm parameters and male fertility potential

Abstract

Purpose: To investigate the relationship between sperm apoptotic biomarkers and patient clinical characteristics, conventional sperm parameters and fertility potential.

Material and methods: Sperm analysis, phospholipid asymmetry, mitochondrial membrane potential (MMP) and DNA denaturation were assessed in 142 males of infertile couples. Seventy-three couples were allocated to the natural conception group, and 55 couples underwent IVF or ICSI.

Results: DNA denaturation correlated positively with age and negatively with testicular volume (TV). MMP correlated negatively with BMI and FSH and positively with TV. Normal viable sperm correlated positively with TV and negatively with age, BMI and FSH. DNA denaturation was associated with a significantly lower natural pregnancy rate (OR 5.4, 95% CI:1.3-22, p = 0.011).

Conclusion: Sperm apoptosis is related to male age, BMI, testicular volume and FSH. Among the apoptotic markers, only DNA denaturation has been found to predict natural pregnancy better than conventional sperm parameters.

Fig. 1

Flow cytometry detection of phosphatidylserine exposure analyzed with annexin V and propidium iodide (PI). Sperm at the final concentration of 1 × 10⁶/mL were incubated with annexin V and 1 μg/mL PI in 100 μL of binding buffer. In the left panel sperm cells were mostly annexin V and PI negative, indicating that they were viable and not undergoing apoptosis. The right panel represents a sample with three populations of cells: normal viable sperm were annexin V and PI negative, early apoptotic sperm were annexin V positive and PI negative, and dead sperm were annexin V ± and PI positive

Fig. 2

Assessment of sperm mitochondrial membrane potential (MMP) measured by means of 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)). Sperm from each fresh sample were incubated with DiOC₆(3). Ten thousand cells were analyzed by FACSCalibur flow cytometer. The left sample has a high percentage of viable sperm cells with normal MMP. The right sample has more cells with lower MMP

Fig. 3

Flow cytometry analysis of sperm DNA denaturation. Sperm cells were stained with acridine orange. The left sample shows mostly native double-stranded DNA in the gate R2. In the right sample we can see two populations of cells: cells with native double-stranded DNA in the gate R2, and cells with denatured single-stranded DNA in the gate R3

Fig. 4

Sperm at the final concentration of 1 × 10⁶/mL were incubated with DiOC₆(3) and 1μg/mL propidium iodide (PI) and analyzed by confocal microscopy. Sperm in the upper left side of the photograph has normal mitochondrial membrane potential (MMP) and is defined as viable sperm with normal MMP. Sperm at the bottom right has low MMP and damaged plasma membrane which is permeable to PI