Dynamic assessment of human sperm DNA damage II: the effect of sperm concentration adjustment during processing

Abstract

Purpose To evaluate the effect of sperm concentration adjustment in human ejaculates on the sperm DNA quality and longevity. Methods Semen samples were obtained from 30 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended in PBS, and the sperm concentration adjusted to 200, 100, 50, 25, 12, and 6 × 10 6 /mL. Each set of samples was incubated at 37 °C for 24 h, and the sperm DNA damage was assessed using the chromatin-dispersion test following 0 h, 2 h, 6 h, and 24 h of incubation. Results Sperm DNA fragmentation (SDF) did not differ between the selected experimental conditions at T0; however, Kaplan-Meier estimates for survival showed significant differences with respect to the dilution and time (all P values were smaller than .001). DNA fragmentation in semen samples adjusted to 200 × 10 6 /mL was approximately 3.3 times higher when compared to samples containing 25 × 10 6 /mL and 3.9 higher in comparison with samples adjusted to 12 × 10 6 /mL following 2 h of in vitro incubation. Although there was evidence of individual variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. Conclusions Incubation of spermatozoa for ART purposes should be done following a concentration adjustment below 25 × 10 6 / mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage

“…With regard to the effect of sperm concentration on DNA fragmentation, we observed that sDFI was higher when sperm concentration increased. It was observed that DNA longevity depends largely on sperm concentration during incubation [ 56 ]. After incubation at 37 °C, the DNA fragmentation rate was greater in samples with high sperm concentration, but it had a significant individual effect [ 56 , 57 ].…”

“…It was observed that DNA longevity depends largely on sperm concentration during incubation [ 56 ]. After incubation at 37 °C, the DNA fragmentation rate was greater in samples with high sperm concentration, but it had a significant individual effect [ 56 , 57 ]. One explanation could be that an increase in oxidative stress (e.g., H 2 O 2 ) occurs when spermatozoa are stored at high concentrations [ 4 , 15 ], and it is known that an increase in oxidative stress induces nuclear and mitochondrial DNA damage [ 58 ].…”

Composition of Stallion Seminal Plasma and Its Impact on Oxidative Stress Markers and Spermatozoa Quality

“…All slides were stained using SYBR Green (2 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA) in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) and a minimum of 300 spermatozoa per sample was scored using an Epifluorescence Leica DMI6000 microscope using a dry ×40 fluorite magnification objective (Leica Microsystems, Wetzlar, Germany). The incidence of DNA fragmented spermatozoa was expressed in percentages (%) [42].…”

Direct but Not Indirect Methods Correlate the Percentages of Sperm With Altered Chromatin to the Intensity of Chromatin Damage

“…Analysis of the halo diameter was used as a quantitative parameter for DNA breaks (chromatin damage intensity), whereas the percentage of sperm with fragmented DNA was determined by classifying 400 sperm haloes using the criteria defined for sperm DNA fragmentation (40).…”