Sperm oxidative damage acquired during seminal plasma removal for assisted reproductive technology is reduced by BGP-15

Abstract

Purpose: Semen manipulation for assisted reproductive technology (ART) causes spermatozoa damage; thus, we investigated the potential of the novel therapeutic BGP-15 to preserve sperm quality during semen washing prior to insemination.

Methods: Donated human ejaculates (N = 40), with or without 10 µM BGP-15, were analyzed for sperm motility, DNA fragmentation, and oxidation. Seminal plasma was removed using different clinical sperm selection methods: simple wash, swim-up, or density gradient centrifugation (DGC), followed by assessment for sperm motility, mitochondrial ROS (mtROS), mitochondrial membrane potential (MMP), and DNA fragmentation and oxidation.

Results: Donated semen samples incubated with BGP-15 had increased sperm motility (+ 15%, p = 0.002) and reduced oxidative DNA damage levels (- 57%, p = 0.03). Samples processed by simple wash had the highest sperm count compared with DGC (+ 55%, p < 0.005) and swim-up (+ 21%, p < 0.0005). Swim-up showed increased vitality compared with DGC (+ 18%, p < 0.001) and simple wash (+ 27%, p < 0.0001), as well as the lowest DNA oxidation levels compared with simple wash - 40%, (p = 0.01) and DGC (- 76%, p < 0.0001). Swim-up also had the lowest mitochondrial membrane potential compared with simple wash and DGC (- 28%, p < 0.03). Comparison between untreated and BGP-15-treated groups for each sperm washing method showed that BGP-15 increased MMP in DGC sperm (+ 11%, p = 0.0006), and reduced DNA fragmentation in washed samples (- 22%, p = 0.03). Moreover, BGP-15 lowered DNA oxidation in all preparation methods: washed (- 48%, p = 0.002), swim-up (- 42%, p = 0.04), and DGC (- 29%, p < 0.0001).

Conclusions: The inclusion of BGP-15 during semen preparation can protect sperm quality and, in the future, may be used clinically to improve sperm selection methods.

Keywords: DNA damage; Male; Reproduction; Semen analysis; Spermatozoa*.

Fig. 1

BGP-15 maintains sperm motility and reduces DNA oxidation in semen samples. A Experimental design simulating clinical waiting times and sperm selection methods prior to ART. Patient semen specimens were deposited and waited for use in IVF/ICSI (time to treat). Discarded semen was split, with gamete media containing or lacking BGP-15 added 1:1 v/v (Incubation time). Initial analyses (motility, DNA fragmentation, and DNA oxidation) were performed on untreated ( −) and BGP-15-treated ( +) samples. Each aliquot was then processed using one of three sperm selection methods: wash (W; sperm cells are separated from seminal plasma components), swim-up (SU; highly motile sperm swim into an overlying gamete media layer), or density gradient centrifugation (DGC; motile, viable sperm are concentrated in the pellet while less dense components remain in upper layers), with or without BGP-15. Selected sperm were assessed for count, motility, viability, membrane fluidity, mitochondrial activity (MMP, mtROS), DNA fragmentation, and DNA oxidation. Analyses in panels B–F refer to the initial analysis of the untreated ( −) and BGP-15-treated ( +) whole semen samples prior to sperm selection. B Sperm motility measured as the percentage of total motile sperm cells. C Relationship between time to treat the sample (time elapsed from ejaculate deposit until BGP-15 treatment) and sperm motility at initial semen analysis. D Relationship between incubation time (total time the ejaculate sample was exposed to BGP-15) and sperm motility at initial analysis. E Sperm DNA fragmentation measured as the percentage of sperm negative for HALO/SCD assay. F Sperm DNA oxidation measured as the percentage of sperm positive for nuclear 8OHdG immunodetection. N = 40 ejaculates. Data shown as mean ± SEM. Statistical analysis was either paired, B, E, F two-tailed Student’s t-test or C, D linear regression analysis. *p < 0.05 and **p < 0.01

Fig. 2

BGP-15 improves sperm motility after wash and increases sperm MMP after DGC. Semen specimens were processed by one of three methods: wash (W), swim-up (SU) and density gradient centrifugation (DGC); in the absence ( −) or presence ( +) of BGP-15. A Sperm motility measured as percentage of motile sperm in washed (W) samples. B Sperm mtROS measured as the percentage of sperm cells positive for MitoSox Red superoxide stain using flow cytometry. C Subpopulation of sperm displaying high MMP, measured as the percentage of sperm cells positive for red fluorescence after JC-1 mitochondrial stain using flow cytometry. D Fluorescence intensity in the high MMP sperm subset, measured as median arbitrary units (AU). A, B N = 38 or C, D N = 30 isolated sperm samples. Data shown as mean ± SEM. Statistical analysis was either paired, A two-tailed Student’s t-test or B–D repeated measures mixed model. Post hoc pairwise comparisons were paired Student’s t-test, either within the same treatment group (*p = 0.05, **p < 0.01, and ***p < 0.001), or between untreated and BGP-15-treated samples (^^^p < 0.001) from the same selection method group

Fig. 3

BGP-15 prevents sperm DNA oxidation during the clinical selection of sperm. Semen specimens were processed by wash (W), swim-up (SU), and density gradient centrifugation (DGC); in the absence ( −) or presence ( +) of BGP-15. A Sperm DNA fragmentation levels measured as the percentage of sperm cells that were negative for HALO/SCD assay. B Sperm DNA oxidation levels measured as the percentage of sperm positive for nuclear 8OHdG immunodetection. N = 38 isolated sperm samples. Data shown as mean ± SEM. Statistical analysis was repeated measures mixed model. Post hoc pairwise comparisons were paired Student’s t-test, either between untreated groups only (*p = 0.05, ****p < 0.0001), or between untreated and BGP-15-treated samples from the same method group (^p < 0.05, ^^p < 0.01, and ^^^^p < 0.0001)