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, 12 (3), 388-399

Improvement of Human Sperm Vacuolization and DNA Fragmentation Co-Cultured With Adipose-Derived Mesenchymal Stem Cell Secretome: In Vitro Effect

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Improvement of Human Sperm Vacuolization and DNA Fragmentation Co-Cultured With Adipose-Derived Mesenchymal Stem Cell Secretome: In Vitro Effect

Robert Bader et al. Int J Stem Cells.

Abstract

Background and objectives: Oxidative stress (OS) is known to be an important factor of male infertility. Adipose-derived mesenchymal stem cells (AD-MSCs) are known to have immune-modulatory and anti-oxidant effects through their secretions, hence raising the idea of their potential benefit to improve sperm parameters. This study aims at investigating the effect of AD-MSCs conditioned medium (CM) on human sperm parameters in the presence and absence of H2O2-induced OS.

Methods and results: Sperm samples were collected from 30 healthy men and divided into two groups: non-stressed and H2O2-stressed. Isolated AD-MSCs from healthy donors undergoing liposuction were cultured and CM was collected at 24, 48 and 72 h. Both sperm groups were cultured with CM and a time course was performed followed by an evaluation of sperm parameters. The incubation of non-stressed and stressed sperm samples with AD-MSCs-CM for 24 h was found to have the optimum impact on sperm vacuolization, DNA fragmentation and OS levels in comparison to other incubation timings, while preserving motility, viability and morphology of cells. Incubation with CM improved all sperm parameters except morphology in comparison to the non-treated group, with the best effect noted with CM collected at 24 h rather than 48 or 72 h for sperm vacuolization and DNA fragmentation. When compared to fresh semen parameters (T0), samples cultured with CM 24 h showed a significant decrease in sperm vacuolization and DNA fragmentation while keeping other parameters stable.

Conclusions: AD-MSCSs-CM improves sperm quality, and hence can be used in treating infertility and subsequently enhancing IVF outcomes.

Keywords: Conditioned medium; Male infertility; Mesenchymal stem cells; Oxidative stress; Sperm.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Immunophenotype and differentiation capacity of human adipose-derived mesenchymal stem cells (AD-MSCs). (A) AD-MSCs have fibroblastic features at passage 1 (P1) (magnification 100×). (B) The flow cytometry histograms for a representative donor are displayed at P1. The percentage of cells stained positive is indicated in the upper right corner of each panel. The green line indicates the positively stained cells, whereas the purple line indicates the isotype-matched monoclonal antibody control. (C) Cells were incubated for 21 days in adipogenic, osteogenic and chondrogenic media. Representative images of AD-MSCs differentiation into (a) adipocytes identified by the presence of intracellular lipid droplets that were confirmed by Oil-Red-Staining, (b) osteocytes characterized by the formation of calcium deposits stained by red alizarin, and (c) chondrocytes which presence was revealed by Acian Blue staining. Scale bar=100 μm.
Fig. 2
Fig. 2
Establishment of the optimum incubation period for sperm parameters improvement. Sperm collected from 30 healthy individuals were incubated for 1 hour in the presence or absence of 10 μM H2O2. After removal of H2O2, stressed and non-stressed sperm were treated with CM collected at 24, 48 and 72 h of conditioning for different time periods (1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). Sperm motility (A, B), vitality (C, D), vacuolization (E, F), DNA fragmentation (G, H), ROS levels (I, J), and morphology (K, L) were assessed and compared to those of sperm cultured with DMEM-F12 medium, considered as the control group. Data are represented as mean±SD. Differences among groups were evaluated using the non-parametric Two Way ANOVA test further evaluated by Tukey test for post hoc pairwise comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for significance between sperm treated with CM 24, 48 or 72 hours and the control group (F12) at a same time-point. ●p<0.05; ●●p<0.01; ●●●p<0.001; ●●●●p<0.0001 represent differences in results observed in sperm treated with CM for different incubation periods as compared to sperm immediately analyzed after collection (0 h incubation time).
Fig. 2
Fig. 2
Establishment of the optimum incubation period for sperm parameters improvement. Sperm collected from 30 healthy individuals were incubated for 1 hour in the presence or absence of 10 μM H2O2. After removal of H2O2, stressed and non-stressed sperm were treated with CM collected at 24, 48 and 72 h of conditioning for different time periods (1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). Sperm motility (A, B), vitality (C, D), vacuolization (E, F), DNA fragmentation (G, H), ROS levels (I, J), and morphology (K, L) were assessed and compared to those of sperm cultured with DMEM-F12 medium, considered as the control group. Data are represented as mean±SD. Differences among groups were evaluated using the non-parametric Two Way ANOVA test further evaluated by Tukey test for post hoc pairwise comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for significance between sperm treated with CM 24, 48 or 72 hours and the control group (F12) at a same time-point. ●p<0.05; ●●p<0.01; ●●●p<0.001; ●●●●p<0.0001 represent differences in results observed in sperm treated with CM for different incubation periods as compared to sperm immediately analyzed after collection (0 h incubation time).
Fig. 3
Fig. 3
Temporal effect of conditioned medium on human sperm motility (A), vitality (B), vacuolization (C), DNA fragmentation (D), ROS levels (E), and morphology (F). Sperm collected from 30 healthy individuals were incubated for 1 hour in the presence or absence of 10 μM H2O2. After removal of H2O2, stressed and non-stressed sperm were incubated in the presence or absence of CM collected at 24, 48 and 72 hours. After 24-hour incubation at 37°C, sperm parameters were assessed and compared to the control group (sperm cultured with DMEM-F12 medium). Differences among groups were evaluated using the non-parametric One Way ANOVA test further tested by Tukey test for post hoc pairwise comparisons. Data are represented as mean±SD. **p<0.01; ***p<0.001; ****p<0.0001 for differences between CM-treated groups and F12. ●p<0.05; ●●●●p<0.0001 for differences between sperm treated with CM 48 h or CM 72 h and sperm treated with CM 24 h.
Fig. 4
Fig. 4
Effect of CM 24 h on human sperm motility (A), vitality (B), DNA fragmentation (C), vacuolization (D), and ROS levels (E). Parameters of stressed and non-stressed samples treated with CM 24 h were assessed and compared to those of fresh semen immediately tested after collection. Differences among groups were evaluated using the non-parametric One Way ANOVA test further tested by Tukey test for post hoc pairwise comparisons. Data are represented as mean±SD. *p<0.05; ***p<0.001; ****p<0.0001.

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References

    1. Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century. Hum Reprod Update. 2015;21:411–426. doi: 10.1093/humupd/dmv016. - DOI - PubMed
    1. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37. doi: 10.1186/s12958-015-0032-1. - DOI - PMC - PubMed
    1. Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health. 2014;32:1–17. doi: 10.5534/wjmh.2014.32.1.1. - DOI - PMC - PubMed
    1. Herrero C, Pérez-Simón JA. Immunomodulatory effect of mesenchymal stem cells. Braz J Med Biol Res. 2010;43:425–430. doi: 10.1590/S0100-879X2010007500033. - DOI - PubMed
    1. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–317. doi: 10.1080/14653240600855905. - DOI - PubMed

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