Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia

Abstract

Background: Leukocytes contribute directly and indirectly to reactive oxygen species (ROS) production. Although leukocytospermia is defined as the presence of ≥ 1 × 106 white blood cells/mL (WBC/mL) in a semen sample, the presence of less than 1×10(6) WBC/mL (low-level leukocytospermia) can still produce a detectable amount of ROS, impairing sperm function and lowering the chances of pregnancy. Our objective was to assess the effect of low-level leukocytospermia on semen quality, ROS levels, and DNA damage in infertile men.

Methods: Semen samples were examined from 472 patients and divided into 3 groups: no seminal leukocytes; group 2, men with low-level leukoctyospermia (0.1-1.0 × 106 WBC/mL); and group 3, frank leukocytospermia, (>1.0 × 106. WBC/mL). Semen analysis, leukoctyospermia, reactive oxygen species and DNA fragmentation was tested.

Results: Conventional semen parameters between the 3 groups were similar. Group 2 patients had significantly higher levels of ROS and sperm DNA fragmentation (1839.65 ± 2173.57RLU/s; DNA damage: 26.47 ± 19.64%) compared with group 1 (ROS: 1101.09 ± 5557.54 RLU/s; DNA damage: 19.89 ± 17.31%) (ROS: p=0.002; DNA damage: p=0.047). There was no significant difference in ROS levels between groups 2 and 3.

Conclusions: Patients presenting with low-level leukocytospermia have seminal oxidative stress. Although these patients are not categorized as leukocytospermic by current World Health Organization (WHO) guidelines, these men may benefit by treatment with antibiotics, testing for bacterial cultures, or antioxidant supplements to reduce ROS-induced sperm DNA fragmentation and improve their chances of fertility. The WHO guidelines for leukocytospermia may need to be revised accordingly.

Figure 1

Flow chart demonstrating inclusion and exclusion criteria. This chart demonstrates that while 472 patients initially presented with infertility, only 211 were included in this study. 22 were excluded due to too few round cells present in their semen, and 239 patients were excluded due to their history of varicocele. Patients included were of reproductive age, and had been deemed infertile between 1–12 years.

Figure 2

Box plot demonstrating minimum, maximum, median, and upper and lower quartiles of ROS data. This box-plot demonstrates the differences in ROS median, range, and upper and lower quartiles. It demonstrates that the median of the leukocytospermic group (median: 1,286.8 RLU/sec) is much higher than both the low leukocytospermic group, and the non-leukocytospermic group. Additionally, the median of the low leukocytospermic group (median: 944.8 RLU/sec) is also much higher than the non-leukocytospermic group (median: 116.7 RLU/sec).

Figure 3

Flow cytometry results for DNA fragmentation. DNA damage was measured by flow cytometry, FITC-deoxyuridine triphosphate (dUTP) substrate was added to the TdT enzyme binding with the free 3′-OH termini of the single – and double strand DNA. DNA damage was measured by gating the population of cells as negative or positive. PI stains total DNA and FITC-dUTP stains apoptotic cells. A: Representative histogram for a sample that is negative for DNA damage and B: Representative histogram for a sample tested positive for DNA damage by flow cytometry.