Nerve growth factor in human semen: Effect of nerve growth factor on the normozoospermic men during cryopreservation process

Abstract

Objectives: Although routinely applied in assisted reproductive technology, human sperm cryopreservation is not a completely successful procedure. Adverse effects of cryopreservation on the fertilization capacity, motility, morphology, and viability of spermatozoa have been proven; cryopreservation has also shown a role in sperm DNA fragmentation and infertility. The post-thaw survival of spermatozoa improved after addition of supplementation of antioxidant molecules to freezing media. Nerve growth factor (NGF) as one of the prosurvival substances has gained great attention in recent years. The aim of this study was the usage of NGF as prosurvival factor after cryopreservation process of human semen samples to assess the motility and viability of sperm, nitric oxide (NO) concentration, and DNA fragmentation in normozoospermic men.

Materials and methods: Semen samples were collected from 25 normozoospermic men and were divided into fresh semen samples as control group, frozen-thawed semen samples without addition of exogenous NGF, and three groups of semen samples cryopreserved with addition of exogenous NGF (0.5, 1, and 5 ng/ml) in freezing medium. Viability was assessed by eosin-negrosin staining technique. Motility was evaluated with inverted microscope. NO concentration and apoptosis content were measured with flow cytometry.

Results: Results showed that exogenous NGF at 0.5 ng/ml could significantly (P-value <0.05) influence viability, motility, nitric oxide, and DNA fragmentation content.

Conclusion: Exogenous NGF as cryoprotectant improved sperm viability and motility, increased intracellular NO concentration, and decreased apoptosis content in normal human spermatozoa.

Keywords: Apoptosis; Cryopreservation; Human sperm; Nerve growth factor; Nitric oxide.

Figure 1

Effect of nerve growth factor on sperm viability in normozoospermic men (a). Human spermatozoa staining with eosin-nigrosin dye assessed by oil immersion light microscopy at ×1000 magnification. Live spermatozoa appeared white whilst dead spermatozoa with disrupted membranes have taken up the eosin stain and appeared red (b). P-values <0.05 were considered significant.*: Significant difference vs. fresh group (P-value <0.05). **: Significant difference vs. frozen-thawed group (P-value <0.05). Error bars: ±1 SE

Figure 2

Effect of nerve growth factor on sperm nitric oxide content in normozoospermic men (a). Dot plot represents total acquired events and final gated population of spermatozoa (b). Histogram of unstaining semen sample (c). Histogram of semen sample, incubated with baseline 4,5-diaminofluorescein-2/diacetate fluorescence (d). P-values <0.05 were considered significant.*: Significant difference vs. fresh group (P-value <0.05). **: Significant difference vs. frozen-thawed group (P-value <0.05). Error bars: ±1 SE

Figure 3

Effect of nerve growth factor on sperm DNA fragmentation in normozoospermic men (a). Dot plot represents total acquired events and final gated population of spermatozoa (b). Histogram of unstaining semen sample (c). Histogram of semen sample, incubated with TUNEL reaction mixture (d). P-values <0.05 were considered significant.*: Significant difference vs. fresh group (P-value <0.05). **: Significant difference vs. frozen-thawed group (P-value <0.05). Error bars: ±1 SE