Flow cytometry analysis of sperm quality in sub‑fertile men

Czech version

Authors: Zuzana Krátká ¹; Štěpánka Luxová ¹; Viktor Vik ²
Authors‘ workplace: Imunologická laboratoř GENNET, s. r. o., Praha ¹; Andrologie a urologie GENNET, s. r. o., Praha ²
Published in: Ces Urol 2017; 21(4): 289-298
Category: Original Articles

Overview

Objective:
Overview of sperm quality data measured by flow cytometry.

Material and method:
800 sub‑fertile men (aged 21–66 years) were treated at GENNET – assisted reproduction centres. 366 patients had normal results and 434 patients had pathological parameters of semen analysis. Flow cytometry analyses of apoptotic sperm were done in all patients. DNA fragmentation rate was measured by TUNEL assay in 65 samples. The acrosome integrity was measured in 213 samples. Two groups of patients were set according to the number of apoptotic sperm in the semen samples – ApoHigh group included samples with > 50 % of apoptotic sperm and ApoLow group included samples with < 50 % of apoptotic sperm.

Results:
16.9 % of normal semen samples were ApoHigh as well as 53.9 % pathological semen. Higher average values of DNA fragmentation were detected in the ApoHigh group than in the ApoLow group both in normal semen (24.0 % vs. 13.5 %) and in pathologic semen too (33.3 % vs. 18.0 %). Higher average values of the acrosome integrity were detected in the ApoHigh group than in the ApoLow group both in normal semen (40.2 % vs. 23.9 %) and in the pathologic samples as well (53.1 % vs. 35.2 %).

Conclusion:
The presence of apoptotic sperm decreases the semen quality. The examination of apoptosis, DNA fragmentation and acrosome integrity together with microscopic semen analyses provide a better diagnostic tool how to identify sub‑fertile men, than simple semen analysis. These tests might be helpful for selection of patients for further uro‑andrological examination and treatment.

Keywords:
Apoptosis, DNA integrity, fertility, men, sperm quality.

Sources

1. Sharlip ID, Jarow JP, Belker AM, et al. Best practice policies for male infertility. Fertil Steril 2002; 77: 873–882.

2. 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

3. WHO World health organization laboratory manual for examination of human semen. 5th ed. Geneve: WHO press 2010.

4. Evenson DP. The sperm chromatin structure assay (SCSA) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Anim Reprod Sci 2016; 169: 56–75.

5. Sakkas D, Ramalingam M, Garrido N, Barratt CLR. Sperm selection in natural conception: what can we learn from mother nature to improve assisted reproduction outcomes? Hum Reprod Update 2015; 21(6): 711–726.

6. Aitken RJ, Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl 2011; 13: 36–42.

7. Cissen M, Wely MV, Scholten I, et al. Measuring Sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta‑analyses. PLoS One 2016; 11(11): e0165125.doi: 10.1371/journal.pone.0165125.

8. Oosterhuis GJ, Mulder AB, Kalsbeek‑Batenburg EK, et al. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril 2000; 74(2): 245–250.

9. Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999; 5(2): 109–115.

10. Vermes I, Haanen C, Steffens‑Nakken H, Reutelingsperger CPM. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184: 39–51.

11. Sergerie M, Laforest G, Bujan L, Bissonnette F, Bleau G. Sperm fragmentation: treashold value in male fertility. Hum Reprod 2005; 20(12): 3446–3451.

12. Sharma RK, Sabanegh E, Mahfouz R, et al. TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 2010; 76(6): 1380–1386.

13. Krátká Z. Kvalita DNA ve spermiích je negativně ovlivněna věkem mužů a je rizikovým faktorem početí. Ces. Gynek. 2017; 82 (6): 490–494.

14. Aitken RJ, Baker MA. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int J Dev Biol 2013; 57: 265–272.

15. Semet M, Paci M, Saïas‑Magnan J, et al. The impact of drugs on male fertility: a review. Andrology 2017; 5(4): 640–663.

16. Vecoli C, Montano L, Andreassi MG. Environmental pollutans: genetic damage and epigenetic changes in male germ cells. Environ Sci Pollut Res 2016; 23: 23339–23348.

17. Aziz N, Said T, Paasch U, Agarwal A. The relationship between human sperm apoptosis, morphology and the sperm deformity index. Hum Reprod 2007; 22(5): 1413–1419.

18. Simon L, Zini A, Dyachenko A, Ciampi A, Careell DT. A systematic review and meta‑analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome. Asian J Androl 2017; 19: 80–90.

19. Novotná B, Svobodová L, Čechová M, et al. Naše první zkušenosti s využitím kometového testu při hodnocení integrity DNA ve spermiích. Ces Urol 2016; 20(4): 317–325.

20. Peknicova J, Chladek D, Hozak P. Monoclonal antibodies against sperm intra‑acrosomal antigens as markers for male infertility diagnostics and estimation of spermatogenesis. AJRI 2005; 53: 42–49.

21. Capkova J, Kubatova A, Ded L, Tepla O, Peknicova J. Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies. Asian J Androl 2016; 18(1): 108–113.

22. Sebkova N, Cerna M, Ded L, Peknicova J, Dvorakova‑Hortova K. The slower the better: how sperm capacitation and acrosome reaction is modified in the presence of estrogens. Reproduction 2012; 143: 297–307.

23. Meseguer M, Santiso R, Harrero S, Remohi J, Fernandesz JL. Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality. Fertil Steril 2011; 95(1): 124–128.

24. Gil M, Sar‑Shalom V, Sivira YM, Carreras R, Checa MA. Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic review and meta‑analysis. J Assist Reprod Genet 2013; 30: 479–485.