Apoptotic M540 bodies present in human semen interfere with flow cytometry-assisted assessment of sperm DNA fragmentation and oxidation
Tóm tắt
The use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. It has been hypothesized that the observed discrepancies could be attributed to the presence of apoptotic bodies that can be labeled with merocyanine 540, the so-called M540 bodies. In order to verify this hypothesis and determine the accuracy of our in-house FC-assisted evaluation of spermatozoa parameters, we used FC to evaluate both the fragmentation of sperm DNA using the TUNEL assay and the oxidation of sperm DNA using the 8-OHdG assay on semen samples with or without M540 bodies. We show that the presence of M540 bodies lead to underestimation of both the level of sperm DNA fragmentation and sperm DNA oxidation when using FC assisted detection systems. We also observed that this situation is particularly pertinent in semen samples classified as abnormal with respect to the routine WHO semen evaluation as they appear to contain more M540 bodies than normal samples. We conclude that M540 bodies interfere with both FC-conducted assays designed to evaluate sperm nuclear/DNA integrity. Exclusion of these contaminants in unprepared semen samples should be performed in order to correctly appreciate the true level of sperm DNA/nuclear damage which is known to be a critical male factor for reproductive success.
Tài liệu tham khảo
World Health Organization, WHO. In: laboratory manual for examination and processing of human semen. Geneva: WHO Press; 2010.
Choucair FB, Rachkidi EG, Raad GC, Saliba EM, Zeidan NS, Jounblat RA, et al. High level of DNA fragmentation in sperm of Lebanese infertile men using sperm chromatin dispersion test. Middle East Fertility Soc J. 2016;21(4):269–76.
Bach PV, Schlegel PN. Sperm DNA damage and its role in IVF and ICSI. Basic Clin Androl. 2016;26(1):1–10.
Aitken RJ, De Iuliis GN, Nixon B. The sins of our forefathers: paternal impacts on de novo mutation rate and development. Annu Rev Genet. 2020;54:1–24.
Aitken RJ, Drevet JR. The importnace of oxidative stress in determining the functionality of mammalian spermatozoa: a double-edged sword. Antioxidants. 2020;9(2):111. https://doi.org/10.3390/antiox9020111.
Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction. 2001;122:497–506.
Aitken RJ, De Iuliis GN. Origins and consequences of DNA damage in male germ cells. Reprod BioMed Online. 2007;14(6):727–33.
Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod. 1999;14:2279–85.
Zini A, Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction. CMAJ. 2006;175(5):495–500.
Sakkas D, Álvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril. 2011;93(4):1027–36.
Drevet JR, Aitken RJ. Oxidation of sperm nucleus in mammals: a physiological necessity to some extent with adverse impacts on oocyte and offspring. Antioxidants. 2020;9(2):95. https://doi.org/10.3390/antiox9020095.
Ringertz NR, Gledhill BL, Darżynkiewicz Z. Changes in deoxyribonucleoprotein during spermiogenesis in the bull: sensitivity of DNA to heat denaturation. Exp Cell Res. 1960;62(1):204–18.
Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl. 2002;23(1):25–43.
Simon L, Emery B, Carrell DT. Sperm DNA fragmentation: consequences for reproduction. Adv Exp Med Biol. 2019;1166:87–105.
Rex AS, Aagaard J, Fedder J. DNA fragmentation in spermatozoa: a historical review. Andrology. 2017;5(4):622–30.
Ribeiro SC, Muratori M, De Geyter M, De Geyter C. TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation. PLoS One. 2017;12(8):e0181802. https://doi.org/10.1371/journnal.pone.0181802.
Muratori M, Forti G, Baldi E. Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labeling. Cytometry Part A: J Int Soc Anal Cytol. 2008;73(9):785–7.
Muratori M, Porazzi I, Luconi M, Marchiani S, Forti G, Baldi E. Annexin V binding and merocyanine staining fail to detect human sperm capacitation. J Androl. 2004;25(5):797–810.
Marchiani S, Tamburino L, Maoggi A, Vannelli GB, Forti G, Baldi E, et al. Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies. Mol Hum Reprod. 2007;13(9):621–31.
Marchiani S, Tamburino L, Forti G, Baldi E, Muratori M. M540 bodies and their impact on flow cytometric analyses of human spermatozoa. Soc Reprod Fertil Suppl. 2007;65:509–14.
Muratori M, Tamburrino L, Marchiani S, Guido C, Forti G, Baldi E. Critical aspects of detection of sperm DNA fragmentation by TUNEL/flow cytometry. Syst Biol Reprod Med. 2010;56(4):277–85.
Marchiani S, Tamburrino L, Giuliano L, Nosi D, Sarli V, Gandini L, Piomboni P, Belmonte G, Forti G, Baldi E, Muratori M. Sumo1-ylation of human spermatozoa and its relationship with semen quality. Int J Androl. 2011;34(6pt1):581–93.
Vorilhon S, Brugnon F, Kocer A, Dollet S, Bourgne C, Berger M, et al. Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay. Hum Reprod. 2018;33(4):553–62.
Gomez-Lopez N, Estrada-Gutierez G, Colin A, Flores-Pliego A, Lores-Escobar X, Oehninger S, et al. The apoptotic pathway in fertile and subfertile men: a case-control and prospective study to examine the impact of merocyanine 540 bodies on jeculated spermatozoa. Fertil Steril. 2013;99(5):1242–8.
Muratori M, Marchiani S, Forti G, Baldi E. Sperm ubiquitination positively correlates to normal morphology in human semen. Hum Reprod. 2005;20(4):1035–43.
Lotti F, Tamburrino L, Marchiani S, Muratori M, Corona G, Fino G, Degl'Innocenti S, Forti G, Maggi M, Baldi E. Semen apoptotic M540 body levels correlate with testis abnormalities: a study in a cohort of infertile subjects. Hum Reprod 2012;27(12):3393–3402.
Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, et al. Investigation on the origin of sperm DNA fragmentation: role of apoptosis, immaturity and oxidative stress. Mol Med. 2015;21(1):109–22.
Fathi Z, Tavalaee M, Kiani A, Deemeh MR, Modaresi M, Nasr-Esfahani MH. Flow cytometry: a novel approach for indirect assessment of protamine deficiency by CMA3 staining, taking into account the presence of M540 or apoptotic bodies. Int J Fertil Steril. 2011;5(3):128–33.
Marchiani S, Tamburrino L, Olivito B, Betti L, Azzari C, Forti G, et al. Characterization and sorting of flow cytometric populations in human semen. Andrology. 2014;2(3):394–401.
Muratori M, Marchiani S, Tamburrino L, Tocci V, Faill P, Forti G, et al. Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters. Hum Reprod. 2008;23(5):1035–43.
Antonouli S, Papatheodorou A, Panagiotidis Y, Petousis S, Prapas N, Nottola SA, et al. The impact of sperm DNA fragmentation on ICSI outcome in cases of donated oocytes. Arch Gyn Obstet. 2019;300(1):207–15.
Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, et al. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl. 2003;49(1):49–55.
Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S. Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet. 2012;29(1):11–4.
Oleszczuk K, Giwercman A, Bungum M. Sperm chromatin structure assay in prediction of in vitro fertilization outcome. Andrology. 2016;4(2):290–6.
Cissen M, Wely MV, Scholten I, Mansell S, Bruin JPD, Mol BW, et al. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis. PLoS One. 2016;11(11):e0165125. https://doi.org/10.1371/journal.pone.0165125.